Patent Application
20220325363
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 22, 2020, is named 53694-730_601_SL.txt and is 1,121,102 bytes in size.
Various communicable diseases can easily spread from an individual or environment to an individual. These diseases may include but are not limited to influenza. Individuals with influenza may have poor outcomes. The detection of the ailments, especially at the early stages of infection, may provide guidance on treatment or intervention to reduce the progression or transmission of the ailment.
In various aspects, the present disclosure provides a microfluidic cartridge for detecting a target nucleic acid comprising: an amplification chamber fluidically connected to a valve; a detection chamber fluidically connected to the valve, wherein the valve is connected to a sample metering channel; a detection reagent chamber fluidically connected to the detection chamber via a resistance channel, the detection reagent chamber comprising a programmable nuclease, a guide nucleic acid, and a labeled detector nucleic acid, wherein the labeled detector nucleic acid is capable of being cleaved upon binding of the guide nucleic acid to a segment of a target nucleic acid.
In some aspects, the sample metering channel controls volumes of liquids dispensed in a channel or chamber. In some aspects, the sample metering channel is fluidically connected to the detection chamber. In some aspects, the resistance channel has a serpentine path, an angular path, or a circuitous path. In some aspects, the valve is a rotary valve, pneumatic valve, a hydraulic valve, an elastomeric valve. In some aspects, the resistance channel is fluidically connected with the valve. In some aspects, the valve comprises casing comprising a “substrate” or an “over-mold.” In some aspects, the valve is actuated by a solenoid. In some aspects, the valve is controlled manually, magnetically, electrically, thermally, by a bistable circuit, with a piezoelectric material, electrochemically, with phase change, rheologically, pneumatically, with a check valve, with capillarity, or any combination thereof. In some aspects, the rotary valve fluidically connects at least 3, at least, 4, or at least 5 chambers.
In some aspects, the microfluidic cartridge further comprises an amplification reagent chamber fluidically connected to the amplification chamber. In some aspects, the microfluidic cartridge further comprises a sample chamber fluidically connected to the amplification reagent chamber. In some aspects, the microfluidic cartridge further comprises a sample inlet connected to the sample chamber. In some aspects, the sample inlet is sealable. In some aspects, the sample inlet forms a seal around the sample.
In some aspects, the sample chamber comprises a lysis buffer. In some aspects, the microfluidic cartridge further comprises a lysis buffer storage chamber fluidically connected to the sample chamber. In some aspects, the lysis buffer storage chamber comprises a lysis buffer. In some aspects, the lysis buffer is a dual lysis/amplification buffer.
In some aspects, the lysis buffer storage chamber is fluidically connected to the sample chamber through a second valve. In some aspects, the sample chamber is fluidically connected to the amplification chamber through the amplification reagent chamber. In some aspects, the sample chamber is fluidically connected to the amplification reagent chamber through the amplification chamber. In some aspects, the microfluidic cartridge is configured to direct fluid bidirectionally between the amplification reagent chamber and amplification chamber. In some aspects, the detection reagent chamber is fluidically connected to the amplification chamber. In some aspects, the amplification chamber is fluidically connected to the detection chamber through the detection reagent chamber. In some aspects, comprising a reagent port above the detection chamber configured to deliver fluid from the detection reagent chamber to the detection chamber. In some aspects, the amplification chamber is fluidically connected to the detection reagent chamber through the detection chamber.
In some aspects, the resistance channel is configured to reduce backflow into the detection chamber and the detection reagent chamber. In some aspects, the sample metering channel is configured to direct a predetermined volume of fluid from the detection reagent chamber to the detection chamber. In some aspects, the amplification chamber and detection chamber are thermally isolated. In some aspects, the detection reagent chamber is fluidically connected to the detection chamber. In some aspects, the detection reagent chamber is fluidically connected to the detection chamber via a second resistance channel. In some aspects, the resistance channel or the second resistance channel is a serpentine resistance channel. In some aspects, the resistance channel or the second resistance channel comprises at least two hairpins. In some aspects, the resistance channel or the second resistance channel comprises at least one, at least 2, at least 3, or at least 4 right angles.
In some aspects, the amplification chamber comprises a sealable sample inlet. In some aspects, the sample inlet is configured to form a seal around a swab. In some aspects, microfluidic cartridge is configured to connect to a first pump to pump fluid from the amplification chamber to the detection chamber. In some aspects, microfluidic cartridge is configured to connect to a second pump to pump fluid from the detection reagent chamber to the detection chamber. In some aspects, first pump or the second pump is a pneumatic pump, a peristaltic pump, a hydraulic pump, or a syringe pump. In some aspects, the amplification chamber is fluidically connected to a port configured to receive pneumatic pressure. In some aspects, the amplification chamber is fluidically connected to the port through a channel. In some aspects, the amplification reagent chamber is connected to a second port configured to receive pneumatic pressure. In some aspects, the amplification reagent chamber is fluidically connected to the second port through a second channel.
In some aspects, the microfluidic cartridge is configured to connect to a third pump to pump fluid from the amplification reagent chamber to the amplification chamber. In some aspects, the third pump is a pneumatic pump, a peristaltic pump, a hydraulic pump, or a syringe pump. In some aspects, the detection reagent chamber is connected to a port configured to receive pneumatic pressure. In some aspects, the detection reagent chamber is fluidically connected to a third port through a third channel.
In some aspects, the microfluidic cartridge is configured to connect to a fourth pump to pump fluid from the detection reagent chamber to the detection chamber. In some aspects, the fourth pump is a pneumatic pump, a peristaltic pump, a hydraulic pump, or a syringe pump.
In some aspects, the microfluidic cartridge further comprises a plurality of ports configured to couple to a gas manifold, wherein the plurality of ports is configured to receive pneumatic pressure. In some aspects, any chamber of the microfluidic cartridge is connected to the plurality of ports. In some aspects, the valve is opened upon application of current electrical signal.
In some aspects, the detection reagent chamber is circular. In some aspects, the detection reagent chamber is elongated. In some aspects, the detection reagent chamber is hexagonal. In some aspects, a region of the resistance channel is molded to direct flow in a direction perpendicular to the net flow direction. In some aspects, a region of the resistance channel is molded to direct flow in a direction perpendicular to the axis defined by two ends of the resistance channel. In some aspects, a region of the resistance channel is molded to direct flow along the z-axis of the microfluidic cartridge. In some aspects, the valve is fluidically connected to two detection chambers via an amplification mix splitter. In some aspects, the valve is fluidically connected to 3, 4, 5, 6, 7, 8, 9, or 10 detection chambers via an amplification mix splitter.
In some aspects, the microfluidic cartridge further comprises a second valve fluidically connected to the detection reagent chamber and the detection chamber. In some aspects, the detection chamber is vented with a hydrophobic PTFE vent. In some aspects, the detection chamber comprises an optically transparent surface.
In some aspects, the amplification chamber is configured to hold from 10 μL to 500 μL of fluid. In some aspects, the amplification reagent chamber is configured to hold from 10 μL to 500 μL of fluid. In some aspects, the microfluidic cartridge is configured to accept from 2 μL to 100 μL of a sample comprising a nucleic acid. In some aspects, the amplification reagent chamber comprises between 5 and 200 μl an amplification buffer. In some aspects, the amplification chamber comprises 45 μ1 amplification buffer. In some aspects, the detection reagent chamber stores from 5 to 200 μl of fluid containing the programmable nuclease, the guide nucleic acid, and the labeled detector nucleic acid.
In some aspects, the microfluidic cartridge comprises 2, 3, 4, 5, 6, 7, or 8 detection chambers. In some aspects, the 2, 3, 4, 5, 6, 7, or 8 detection chambers are fluidically connected to a single sample chamber. In some aspects, the detection chamber holds up to 100 μL, 200 μL, 300 μL, or 400 μL of fluid.
In some aspects, the microfluidic cartridge comprises 5-7 layers. In some aspects, the microfluidic cartridge comprises layers as shown in
In some aspects, the microfluidic cartridge further comprises a sliding valve. In some aspects, the sliding valve connects the amplification reagent chamber to the amplification chamber. In some aspects, the sliding valve connects the amplification chamber to the detection reagent chamber. In some aspects, the sliding valve connects the amplification reagent chamber to the detection chamber.
In various aspects, the present disclosure provides a manifold configured to accept the microfluidic cartridge. In some aspects, the manifold comprises a pump configured to pump fluid into the detection chamber, an illumination source configured to illuminate the detection chamber, a detector configured to detect a detectable signal produced by the labeled detector nucleic acid, and a heater configured to heat the amplification chamber. In some aspects, the manifold further comprises a second heater configured to heat the detection chamber.
In some aspects, the illumination source is a broad spectrum light source. In some aspects, the illumination source light produces an illumination with a bandwidth of less than 5 nm. In some aspects, the illumination source is a light emitting diode. In some aspects, the light emitting diode produces white light, blue light, or green light.
In some aspects, the detectable signal is light. In some aspects, the detector is a camera or a photodiode. In some aspects, the detector has a detection bandwidth of less than 100 nm, less than 75 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, less than 10 nm, or less than 5 nm.
In some aspects, the manifold further comprises an optical filter configured to be between the detection chamber and the detector. In some aspects, the amplification chamber comprises amplification reagents. In some aspects, the amplification reagent chamber comprises amplification reagents. In some aspects, the amplification reagents comprise a primer, a polymerase, dNTPs, an amplification buffer. In some aspects, the amplification chamber comprises a lysis buffer. In some aspects, the amplification reagent chamber comprises a lysis buffer. In some aspects, the amplification reagents comprise a reverse transcriptase. In some aspects, the amplification reagents comprise reagents for thermal cycling amplification. In some aspects, the amplification reagents comprise reagents for isothermal amplification. In some aspects, the amplification reagents comprise reagents for transcription mediated amplification (TMA), helicase dependent amplification (HDA), circular helicase dependent amplification (cHDA), strand displacement amplification (SDA), loop mediated amplification (LAMP), exponential amplification reaction (EXPAR), rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence based amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), or improved multiple displacement amplification (IMDA). In some aspects, the amplification reagents comprise reagents for loop mediated amplification (LAMP).
In some aspects, the lysis buffer and the amplification buffer are a single buffer. In some aspects, the lysis buffer storage chamber comprises a lysis buffer. In some aspects, the lysis buffer has a pH of from pH 4 to pH 5.
In some aspects, the microfluidic cartridge further comprises reverse transcription reagents. In some aspects, the reverse transcription reagents comprise a reverse transcriptase, a primer, and dNTPs. In some aspects, the programmable nuclease comprises an RuvC catalytic domain. In some aspects, the programmable nuclease is a type V CRISPR/Cas effector protein. In some aspects, the type V CRISPR/Cas effector protein is a Cas12 protein. In some aspects, the Cas12 protein comprises a Cas12a polypeptide, a Cas12b polypeptide, a Cas12c polypeptide, a Cas12d polypeptide, a Cas12e polypeptide, a C2c4 polypeptide, a C2c8 polypeptide, a C2c5 polypeptide, a C2c10 polypeptide, and a C2c9 polypeptide. In some aspects, the Cas12 protein has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 27-SEQ ID NO: 37. In some aspects, the Cas12 protein is selected from SEQ ID NO: 27-SEQ ID NO: 37.
In some aspects, the type V CRIPSR/Cas effector protein is a Cas14 protein. In some aspects, the Cas14 protein comprises a Cas14a polypeptide, a Cas14b polypeptide, a Cas14c polypeptide, a Cas14d polypeptide, a Cas14e polypeptide, a Cas 14f polypeptide, a Cas14g polypeptide, a Cas14h polypeptide, a Cas14i polypeptide, a Cas14j polypeptide, or a Cas14k polypeptide. In some aspects, the Cas14 protein has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 38-SEQ ID NO: 129. In some aspects, the Cas14 protein is selected from SEQ ID NO: 38-SEQ ID NO: 129.
In some aspects, the type V CRIPSR/Cas effector protein is a CasΦ protein. In some aspects, the CasΦ protein has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 274-SEQ ID NO: 321. In some aspects, the Case protein is selected from SEQ ID NO: 274-SEQ ID NO: 321.
In some aspects, microfluidic cartridge further provides one or more chambers for in vitro transcribing amplified coronavirus target nucleic acid. In some aspects, the in vitro transcribing comprises contacting the amplified coronavirus target nucleic acid to reagents for in vitro transcription. In some aspects, the reagents for in vitro transcription comprise an RNA polymerase, NTPs, and a primer.
In some aspects, the programable nuclease comprises a HEPN cleaving domain. In some aspects, the programmable nuclease is a type VI CRISPR/Cas effector protein. In some aspects, the type VI CRISPR/Cas effector protein is a Cas13 protein. In some aspects, the Cas13 protein comprises a Cas13a polypeptide, a Cas13b polypeptide, a Cas13c polypeptide, a Cas13c polypeptide, a Cas13d polypeptide, or a Cas13e polypeptide. In some aspects, the Cas13 protein has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NOs: 130-SEQ ID NO: 137. In some aspects, the Cas13 protein is selected from SEQ ID NOs: 130-SEQ ID NO: 137.
In some aspects, the target nucleic acid is from a virus. In some aspects, the virus comprises a respiratory virus. In some aspects, the respiratory virus is an upper respiratory virus. In some aspects, the virus comprises an influenza virus. In some aspects, the virus comprises a coronavirus.
In some aspects, the coronavirus target nucleic acid is from SARS-CoV-2. In some aspects, the coronavirus target nucleic acid is from an N gene, an E gene, or a combination thereof. In some aspects, the coronavirus target nucleic acid has a sequence of any one of SEQ ID NO: 333-SEQ ID NO: 338. In some aspects, the influenza virus comprises an influenza A virus, influenza B virus, or a combination thereof. In some aspects, the plurality of target sequences comprises sequences from influenza A virus, influenza B virus, and a third pathogen.
In some aspects, the guide nucleic acid is a guide RNA. In some aspects, the guide nucleic acid has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identify to any one of SEQ ID NO: 323-SEQ ID NO: 328. In some aspects, the guide nucleic acid is selected from any one of SEQ ID NO: 323-SEQ ID NO: 328. In some aspects, the microfluidic cartridge comprises a control nucleic acid. In some aspects, the control nucleic acid is in the detection chamber. In some aspects, the control nucleic acid is RNaseP. In some aspects, the control nucleic acid has a sequence of SEQ ID NO: 379.
In some aspects, the guide nucleic acid has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identify to any one of SEQ ID NO: 330-SEQ ID NO: 332. In some aspects, the guide nucleic acid is selected from any one of SEQ ID NO: 330-SEQ ID NO: 332. In some aspects, the guide nucleic acid targets a plurality of target sequences.
In some aspects, the microfluidic cartridge comprises a plurality of guide sequences tiled against a virus. In some aspects, the labeled detector nucleic acid comprises a single stranded reporter comprising a detection moiety. In some aspects, the detection moiety is a fluorophore, a FRET pair, a fluorophore/quencher pair, or an electrochemical reporter molecule. In some aspects, the electrochemical reporter molecule comprises a species shown in
In various aspects, the present disclosure provides a method of detecting a target nucleic acid, the method comprising: providing a sample from a subject; adding the sample to a microfluidic cartridge; correlating a detectable signal to the presence or absence of a target nucleic acid; and optionally quantifying the detectable signal, thereby quantifying an amount of the target nucleic acid present in the sample.
In some aspects, a microfluidic cartridge may be used in a method for detecting a target nucleic acid. In some aspects, a system may be used in a method for detecting a targeting nucleic acid. In some aspects, a programmable nuclease may be used in a method for detecting a target nucleic acid. In some aspects, a composition may be used in a method for detecting a target a nucleic acid. In some aspects, a DNA-activated programmable RNA nuclease may be used in a method for assaying for a target deoxyribonucleic acid from a virus in a sample. In some aspects, a DNA-activated programmable RNA nuclease may be used in a method of assaying for a target ribonucleic acid from a virus in a sample. In some aspects, a programmable nuclease may be used in a method for detecting a target nucleic acid in a sample.
In various aspects, the present disclosure provides a system for detecting a target nucleic acid, said system comprising: a guide nucleic acid targeting a target sequence from a virus; a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target sequence; and a reporter, wherein the reporter is capable of being cleaved by the activated nuclease, thereby generating a detectable signal.
In some aspects, the reporter comprises a single stranded reporter comprising a detection moiety. In some aspects, the virus comprises an influenza virus. In some aspects, the influenza virus comprises an influenza A virus, influenza B virus, or a combination thereof. In some aspects, the virus comprises a respiratory virus. In some aspects, the respiratory virus is an upper respiratory virus. In some aspects, the guide nucleic acid targets a plurality of target sequences.
In some aspects, the system comprises a plurality of guide sequences tiled against the virus. In some aspects, the plurality of target sequences comprises sequences from influenza A virus, influenza B virus, and a third pathogen. In some aspects, the single stranded reporter comprises the detection moiety at the 5′ end. In some aspects, the single stranded reporter comprises a biotin-dT/FAM moiety or a biotin-dT/ROX moiety. In some aspects, the single stranded reporter comprises a chemical functional handle at the 3′end capable of being conjugated to a substrate.
In some aspects, the substrate is a magnetic bead. In some aspects, the substrate is a surface of a reaction chamber. In some aspects, downstream of the reaction chamber is a test line. In some aspects, the test line comprises a streptavidin. In some aspects, downstream of the test line is a flow control line. In some aspects, the flow control line comprises an anti-IgG antibody. In some aspects, the anti-IgG antibody comprises an anti-rabbit IgG antibody.
In some aspects, the activated nuclease is capable of cleaving the single stranded reporter and releases the biotin-dT/FAM moiety or the biotin-dT/ROX moiety. In some aspects, the biotin-dT/FAM moiety is capable of binding the streptavidin at the test line. In some aspects, the reporter is an electroactive reporter. In some aspects, the electroactive reporter comprises biotin and methylene blue. In some aspects, the reporter is an enzyme-nucleic acid. In some aspects, the enzyme-nucleic acid is an invertase enzyme. In some aspects, an enzyme of the enzyme-nucleic acid is a sterically hindered enzyme.
In some aspects, upon cleavage of a nucleic acid of the enzyme-nucleic acid, the enzyme is functional. In some aspects, the detectable signal is a colorimetric signal, a fluorescence signal, an amperometric signal, or a potentiometric signal.
In various aspects, the present disclosure provides a method of detecting a target nucleic acid in a sample comprising: contacting the sample with a guide nucleic acid targeting a target sequence; a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target sequence; and a reporter, wherein the reporter is capable of being cleaved by the activated nuclease, thereby generating a detectable signal.
In some aspects, the target nucleic acid is from an exogenous pathogen. In some aspects, the exogenous pathogen comprises a virus. In some aspects, the virus comprises an influenza virus. In some aspects, the influenza virus comprises an influenza A virus, influenza B virus, or a combination thereof. In some aspects, the virus comprises a respiratory virus. In some aspects, the respiratory virus is an upper respiratory virus.
In some aspects, the detectable signal indicates presence of the virus in the sample. In some aspects, the method further comprises diagnosing a subject from which the sample was taken with the virus. In some aspects, the subject is a human. In some aspects, the sample is a buccal swab, a nasal swab, or urine. In some aspects, the reporter comprises a single stranded reporter comprising a detection moiety. In some aspects, the guide nucleic acid targets a plurality of target sequences.
In some aspects, the system comprises a plurality of guide sequences tiled against the virus. In some aspects, the plurality of target sequences comprises sequences from influenza A virus, influenza B virus, and a third pathogen. In some aspects, the single stranded reporter comprises the detection moiety at the 5′ end. In some aspects, the single stranded reporter comprises a biotin-dT/FAM moiety or a biotin-dT/ROX moiety. In some aspects, the single stranded reporter comprises a chemical functional handle at the 3′end capable of being conjugated to a substrate. In some aspects, the substrate is a magnetic bead.
In some aspects, the substrate is a surface of a reaction chamber. In some aspects, downstream of the reaction chamber is a test line. In some aspects, the test line comprises a streptavidin. In some aspects, downstream of the test line is a flow control line. In some aspects, the flow control line comprises an anti-IgG antibody. In some aspects, the anti-IgG antibody comprises an anti-rabbit IgG antibody.
In some aspects, the activated nuclease is capable of cleaving the single stranded reporter and releases the biotin-dT/FAM moiety or the biotin-dT/ROX moiety. In some aspects, the biotin-dT/FAM moiety is capable of binding the streptavidin at the test line. In some aspects, the reporter is an electroactive reporter. In some aspects, the electroactive reporter comprises biotin and methylene blue. In some aspects, the reporter is an enzyme-nucleic acid. In some aspects, the enzyme-nucleic acid is an invertase enzyme. In some aspects, an enzyme of the enzyme-nucleic acid is a sterically hindered enzyme. In some aspects, upon cleavage of a nucleic acid of the enzyme-nucleic acid, the enzyme is functional. In some aspects, the detectable signal is a colorimetric signal, a fluorescence signal, an amperometric signal, or a potentiometric signal. In some aspects, in any of the above systems, the respiratory virus is a lower respiratory virus. In some aspects, in any of the above methods, the respiratory virus is a lower respiratory virus.
In some aspects, a composition comprises a DNA-activated programmable RNA nuclease; and a guide nucleic acid comprising a segment that is reverse complementary to a segment of a target deoxyribonucleic acid, wherein the DNA-activated programmable RNA nuclease binds to the guide nucleic acid to form a complex. In some aspects, the composition further comprises an RNA reporter. In some aspects, the composition further comprises the target deoxyribonucleic acid from a virus. In some aspects, the target deoxyribonucleic acid is an amplicon of a nucleic acid. In some aspects, wherein the nucleic acid is a deoxyribonucleic acid or a ribonucleic acid. In some aspects, the DNA-activated programmable RNA nuclease is a Type VI CRISPR/Cas enzyme. In some aspects, the DNA-activated programmable RNA nuclease is a Cas13. In some aspects, the DNA-activated programmable RNA nuclease is a Cas13a. In some aspects, the Cas13a is Lbu-Cas13a or Lwa-Cas13a. In some aspects, the composition has a pH from pH 6.8 to pH 8.2. In some aspects, the target deoxyribonucleic acid lacks a guanine at the 3′ end. In some aspects, the target deoxyribonucleic acid is a single-stranded deoxyribonucleic acid. In some aspects, the composition further comprises a support medium. In some aspects, the composition further comprises a lateral flow assay device. In some aspects, the composition further comprises a device configured for fluorescence detection. In some aspects, the composition further comprises a second guide nucleic acid and a DNA-activated programmable DNA nuclease, wherein the second guide nucleic acid comprises a segment that is reverse complementary to a segment of a second target deoxyribonucleic acid comprising a guide nucleic acid. In some aspects, the composition further comprises a DNA reporter. In some aspects, the DNA-activated programmable DNA nuclease is a Type V CRISPR/Cas enzyme. In some aspects, the DNA-activated programmable DNA nuclease is a Cas12. In some aspects, the Cas12 is a Cas12a, Cas12b, Cas12c, Cas12d, or Cas12e. In some aspects, the DNA-activated programmable DNA nuclease is a Cas14. In some aspects, the Cas14 is a Cas 14a, Cas14b, Cas14c, Cas 14d, Cas 14e, Cas 14f, Cas 14g, or Cas 14h.
In some aspects, a method of assaying for a target deoxyribonucleic acid from a virus in a sample comprises contacting the sample to a complex comprising a guide nucleic acid and a DNA-activated programmable RNA nuclease, wherein the guide nucleic acid comprises a segment that is reverse complementary to a segment of the target deoxyribonucleic acid, and
assaying for a signal produced by cleavage of at least some RNA reporters of a plurality of RNA reporters. In some aspects, a method of assaying for a target ribonucleic acid from a virus in a sample comprises: amplifying a nucleic acid in a sample to produce a target deoxyribonucleic acid; contacting the target deoxyribonucleic acid to a complex comprising a guide nucleic acid and a DNA-activated programmable RNA nuclease, wherein the guide nucleic acid comprises a segment that is reverse complementary to a segment of the target deoxyribonucleic acid, and assaying for a signal produced by cleavage of at least some RNA reporters of a plurality of RNA reporters. In some aspects, the DNA-activated programmable RNA nuclease is a Type VI CRISPR nuclease. In some aspects, the DNA-activated programmable RNA nuclease is a Cas13. In some aspects, the Cas13 is a Cas13a. In some aspects, the Cas13a is Lbu-Cas13a or Lwa-Cas13a. In some aspects, cleavage of the at least some RNA reporters of the plurality of reporters occurs from pH 6.8 to pH 8.2. In some aspects, the target deoxyribonucleic acid lacks a guanine at the 3′ end. In some aspects, the target deoxyribonucleic acid is a single-stranded deoxyribonucleic acid. In some aspects, the target deoxyribonucleic acid is an amplicon of a ribonucleic acid. In some aspects, the target deoxyribonucleic acid or the ribonucleic acid is from an organism. In some aspects, the organism is a virus, bacteria, plant, or animal. In some aspects, the target deoxyribonucleic acid is produced by a nucleic acid amplification method. In some aspects, the nucleic acid amplification method is isothermal amplification. In some aspects, the nucleic acid amplification method is thermal amplification. In some aspects, the nucleic acid amplification method is recombinase polymerase amplification (RPA), transcription mediated amplification (TMA), strand displacement amplification (SDA), helicase dependent amplification (HDA), loop mediated amplification (LAMP), rolling circle amplification (RCA), single primer isothermal amplification (SPIA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), or improved multiple displacement amplification (IMDA), or nucleic acid sequence-based amplification (NASBA). In some aspects, the signal is fluorescence, luminescence, colorimetric, electrochemical, enzymatic, calorimetric, optical, amperometric, or potentiometric. In some aspects, the method further comprises contacting the sample to a second guide nucleic acid and a DNA-activated programmable DNA nuclease, wherein the second guide nucleic acid comprises a segment that is reverse complementary to a segment of a second target deoxyribonucleic acid comprising a guide nucleic acid. In some aspects, the method further comprises assaying for a signal produced by cleavage of at least some DNA reporters of a plurality of DNA reporters. In some aspects, the DNA-activated programmable DNA nuclease is a Type V CRISPR nuclease. In some aspects, the DNA-activated programmable DNA nuclease is a Cas12. In some aspects, the Cas12 is a Cas12a, Cas12b, Cas12c, Cas12d, or Cas12e. In some aspects, the DNA-activated programmable DNA nuclease is a Cas14. In some aspects, the Cas14 is a Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, or Cas14h. In some aspects, the guide nucleic acid comprises a crRNA. In some aspects, the guide nucleic acid comprises a crRNA and a tracrRNA. In some aspects, the signal is present prior to cleavage of the at least some RNA reporters. In some aspects, the signal is absent prior to cleavage of the at least some RNA reporters. In some aspects, the sample comprises blood, serum, plasma, saliva, urine, mucosal sample, peritoneal sample, cerebrospinal fluid, gastric secretions, nasal secretions, sputum, pharyngeal exudates, urethral or vaginal secretions, an exudate, an effusion, or tissue. In some aspects, the method is carried out on a support medium. In some aspects, the method is carried out on a lateral flow assay device. In some aspects, the method is carried out on a device configured for fluorescence detection.
In various aspects, the present disclosure provides a method of designing a plurality of primers for amplification of a target nucleic acid, the method comprising: providing a target nucleic acid, herein a guide nucleic acid hybridizes to the target nucleic acid and wherein at least 60% of a sequence of the target nucleic acid is between an F1c region and a B1 region or between an F1 and a B1c region; and designing the plurality of primers comprising: i) a forward inner primer comprising a sequence of the F1c region 5′ of a sequence of an F2 region; ii) a backward inner primer comprising a sequence of the B1c region 5′ of a sequence of a B2 region; iii) a forward outer primer comprising a sequence of an F3 region; and iv) a backward outer primer comprising a sequence of a B3 region.
In various aspects, the present disclosure provides a method of detecting a target nucleic acid in a sample, the method comprising: contacting the sample to: a plurality of primers comprising: i) a forward inner primer comprising a sequence corresponding to an F1c region 5′ of a sequence corresponding to an F2 region; ii) a backward inner primer comprising a sequence corresponding to a B1c region 5′ of a sequence corresponding to a B2 region; iii) a forward outer primer comprising a sequence corresponding to an F3 region; and iv) a backward outer primer comprising a sequence corresponding to a B3 region; a guide nucleic acid, wherein the guide nucleic acid hybridizes to the target nucleic acid and wherein at least 60% of a sequence of the target nucleic acid is between the F1c region and a B1 region or between an F1 region and the B1c region; a reporter; and a programmable nuclease that cleaves the reporter when complexed with the guide nucleic acid; and
measuring a detectable signal produced by cleavage of the reporter, wherein the measuring provides for detection of the target nucleic acid in the sample.
In some aspects, the sequence between the F1c region and the B1 region or the sequence between the B1c region and the F1 region is at least 50% reverse complementary to the guide nucleic acid sequence. In some aspects, the guide nucleic acid sequence is reverse complementary to no more than 50% of the forward inner primer, the backward inner primer, or a combination thereof. In some aspects, the guide nucleic acid does not hybridize to the forward inner primer and the backward inner primer.
In some aspects, a protospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of the target nucleic acid. In some aspects, a protospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of the B1 region and 5′ of the F1c region or the protospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of the Fl region and 5′ of the B1c region. In some aspects, the 3′ end of the target nucleic acid is 5′ of the 5′ end of the F3c region or the 3′ end of the target nucleic acid is 5′ of the 5′ end of the B3c region. In some aspects, the 3′ end of the target nucleic acid is 5′ of the 5′ end of the F2c region or 3′ end of the target nucleic acid is 5′ of the 5′ end of the B2c region. In some aspects, the target nucleic acid is between the F1c region and the B1 region and the 3′ end of the target nucleic acid is 5′ of the 3′ end of the F2c region, or wherein the target nucleic acid is between the B1c region and the F1 region and the 3′ end of the target nucleic acid is 5′ of the 3′ end of the B2c region.
In some aspects, the guide nucleic acid has a sequence reverse complementary to no more than 50% of the forward inner primer, the backward inner primer, the forward outer primer, the backward outer primer, or any combination thereof. In some aspects, the guide nucleic acid sequence does not hybridize to the forward inner primer, the backward inner primer, the forward outer primer, the backward outer primer, or any combination thereof.
In some aspects, the guide nucleic acid sequence has a sequence reverse complementary to no more than 50% of a sequence of an F3c region, an F2c region, the F1c region, the B1c region, an B2c region, an B3c region, or any combination thereof. In some aspects, the guide nucleic acid sequence does not hybridize to a sequence of an F3c region, an F2c region, the F1c region, the B1c region, an B2c region, an B3c region, or any combination thereof.
In various aspects, the present disclosure provides a method of designing a plurality of primer for amplification of a target nucleic acid, the method comprising: providing the target nucleic acid comprising a sequence between a B2 region and a B1 region or between an F2 region and an F1 region that hybridizes to a guide nucleic acid; and designing the plurality of primers comprising: i) a forward inner primer comprising a sequence of the F1c region 5′ of a sequence of an F2 region; ii) a backward inner primer comprising a sequence of the B1c region 5′ of a sequence of a B2 region; iii) a forward outer primer comprising a sequence of an F3 region; and iv) a backward outer primer comprising a sequence of a B3 region.
In various aspects, the present disclosure provides a method of designing a plurality of primer for amplification of a target nucleic acid, the method comprising: providing the target nucleic acid comprising a sequence between a F1c region and an F2c region or between a B1c region and a B2c region that hybridizes to a guide nucleic acid; and designing the plurality of primers comprising: i) a forward inner primer comprising a sequence of the F1c region 5′ of a sequence of an F2 region; ii) a backward inner primer comprising a sequence of the B1c region 5′ of a sequence of a B2 region; iii) a forward outer primer comprising a sequence of an F3 region; and iv) a backward outer primer comprising a sequence of a B3 region.
In various aspects, the present disclosure provides a method of detecting a target nucleic acid in a sample, the method comprising: contacting the sample to: a plurality of primers comprising: i) a forward inner primer comprising a sequence corresponding to an F1c region 5′ of a sequence corresponding to an F2 region; ii) a backward inner primer comprising a sequence corresponding to a B1c region 5′ of a sequence corresponding to a B2 region; iii) a forward outer primer comprising a sequence corresponding to an F3 region; and iv) a backward outer primer comprising a sequence corresponding to a B3 region; a guide nucleic acid, wherein the target nucleic acid comprises a sequence between a B2 region and a B1 region or between the F2 region and an F1 region that hybridizes to the guide nucleic acid; a reporter; and a programmable nuclease that cleaves the reporter when complexed with the guide nucleic acid; and measuring a detectable signal produced by cleavage of the reporter, wherein the measuring provides for detection of the target nucleic acid in the sample.
In various aspects, the present disclosure provides a method of detecting a target nucleic acid in a sample, the method comprising: contacting the sample to: a plurality of primers comprising: i) a forward inner primer comprising a sequence corresponding to an F1c region 5′ of a sequence corresponding to an F2 region; ii) a backward inner primer comprising a sequence corresponding to a B1c region 5′ of a sequence corresponding to a B2 region; iii) a forward outer primer comprising a sequence corresponding to an F3 region; and iv) a backward outer primer comprising a sequence corresponding to a B3 region; a guide nucleic acid, wherein the target nucleic acid comprises a sequence between the F1c region and an F2c region or between the B1c region and a B2c region that hybridizes to the guide nucleic acid; a reporter; and a programmable nuclease that cleaves the reporter when complexed with the guide nucleic acid; and measuring a detectable signal produced by cleavage of the reporter, wherein the measuring provides for detection of the target nucleic acid in the sample.
In some aspects, a protospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of the B2 region and 5′ of the B1 region or the protospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of the F2 region and 5′ of the F1 region. In some aspects, a protospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of the B1c region and 5′ of the B2c region or the protospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of the F1c region and 5′ of the F2c region.
In some aspects, a protospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of the target nucleic acid. In some aspects, the PAM and the PFS are 5′ of the 5′ end of the F1c region, 5′ of the 5′ end of the B1c region, 3′ of the 3′ end of the F3 region, 3′ of the 3′ end of the B3 region, 3′ of the 3′ end of the F2 region, 3′ of the 3′ end of the B2 region, or any combination thereof
In some aspects, the PAM and the PFS do not overlap the F2 region, the B3 region, the F1c region, the F2 region, the B1c region, the B2 region, or any combination thereof. In some aspects, the PAM and the PFS do not hybridize to the forward inner primer, the backward inner primer, the forward outer primer, the backward outer primer, or any combination thereof.
In some aspects, the plurality of primers further comprises a loop forward primer. In some aspects, the plurality of primers further comprises a loop backward primer. In some aspects, the loop forward primer is between an F1c region and an F2c region. In some aspects, the loop backward primer is between a B1c region and a B2c region.
In some aspects, the target nucleic acid comprises a single nucleotide polymorphism (SNP). In some aspects, the single nucleotide polymorphism (SNP) comprises a HERC2 SNP. In some aspects, the single nucleotide polymorphism (SNP) is associated with an increased risk or decreased risk of cancer. In some aspects, the target nucleic acid comprises a single nucleotide polymorphism (SNP), and wherein the detectable signal is higher in the presence of a guide nucleic acid that is 100% complementary to the target nucleic acid comprising the single nucleotide polymorphism (SNP) than in the presence of a guide nucleic acid that is less than 100% complementary to the target nucleic acid comprising the single nucleotide polymorphism (SNP).
In some aspects, the plurality of primers and the guide nucleic acid are present together in a sample comprising the target nucleic acid. In some aspects, the contacting the sample to the plurality of primers results in amplifying the target nucleic acid. In some aspects, the amplifying and the contacting the sample to the guide nucleic acid occurs at the same time. In other aspects, the amplifying and the contacting the sample to the guide nucleic acid occur at different times. In some aspects, the method further comprises providing a polymerase, a dATP, a dTTP, a dGTP, a dCTP, or any combination thereof.
In some aspects, the target nucleic acid is from a virus. In some aspects, the virus comprises an influenza virus, respiratory syncytial virus, or a combination thereof. In further aspects, the influenza virus comprises an influenza A virus, influenza B virus, or a combination thereof. In some aspects, the virus comprises a respiratory virus. In further aspects, the respiratory virus is an upper respiratory virus.
In some aspects, the system further comprises a forward inner primer, a backward inner primer, a forward outer primer, a backward outer primer, a loop forward primer, a loop backward primer, or any combination thereof. In some aspects, method further comprising contacting the sample with a forward inner primer, a backward inner primer, a forward outer primer, a backward outer primer, a loop forward primer, a loop backward primer, or any combination thereof. In some aspects, method further comprising amplifying the target deoxyribonucleic acid with a forward inner primer, a backward inner primer, a forward outer primer, a backward outer primer, a loop forward primer, a loop backward primer, or any combination thereof. In some aspects, the amplifying comprises contacting the sample to a forward inner primer, a backward inner primer, a forward outer primer, a backward outer primer, a loop forward primer, a loop backward primer, or any combination thereof.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:
The present disclosure provides various devices, systems, fluidic devices, and kits for rapid lab tests, which may quickly assess whether a target nucleic acid is present in a sample by using a programmable nuclease that can interact with functionalized surfaces of the fluidic systems to generate a detectable signal. In particular, provided herein are various devices, systems, fluidic devices, and kits for rapid lab tests, which may quickly assess whether a target nucleic acid is present in a biological sample. The target nucleic acid may be from a virus. For example, the devices, systems fluidic devices, and kits for rapid lab tests disclosed herein may assess whether a target nucleic acid from a strain of influenza virus is present in a sample. The influenza can be influenza A or influenza B. The virus may be a coronavirus. The compositions and methods provided herein disclose programmable nucleases that can be used in the systems, fluidic devices, and kits provided herein to detect target nucleic acids from influenza or another virus, for example another respiratory virus (e.g., coronavirus). In some embodiments, the target nucleic acids can be from an upper respiratory tract virus. In some embodiments, provided herein are devices, systems, fluidic devices, and kits that can perform multiplexed detection of more than one unique sequence of target nucleic acids. For example, the devices, systems, fluidic devices, kits, and programmable nucleases provided herein can be used for multiplexed detection of target nucleic acids from one or more than viruses. In particular embodiments, the devices, systems, fluidic devices, kits, and programmable nucleases provided herein can be used for multiplexed detection of influenza A and influenza B. In some embodiments, devices, systems, fluidic devices, kits, and programmable nucleases provided herein can be used for multiplexed detection of influenza A, influenza B, and one or more other viruses (e.g., coronavirus, RSV or another respiratory virus, such as an upper respiratory tract virus).
The systems and programmable nucleases disclosed herein can be used as a companion diagnostic with any of the diseases disclosed herein (e.g., RSV, sepsis, flu), or can be used in reagent kits, point-of-care diagnostics, or over-the-counter diagnostics. The systems may be used as a point of care diagnostic or as a lab test for detection of a target nucleic acid and, thereby, detection of a condition in a subject from which the sample was taken. The systems may be used to determine the presence or absence of a gene of interest (e.g., a gene associated with a disease state) in a subject from which the sample was taken. The systems may be used to determine the presence or absence of a pathogen (e.g., a virus or bacterium) in a subject from which the sample was taken. The systems may be used in various sites or locations, such as in laboratories, in hospitals, in physician offices/laboratories (POLs), in clinics, at remotes sites, or at home. Sometimes, the present disclosure provides various devices, systems, fluidic devices, and kits for consumer genetic use or for over the counter use.
Described herein are devices, systems, fluidic devices, kits, and methods for detecting the presence of a target nucleic acid in a sample. A target nucleic acid may be a gene, or a portion of a gene, associated with a disease state. A target nucleic acid may be a nucleic acid from a pathogen (e.g., a virus or a bacterium). The devices, systems, fluidic devices, kits, and methods for detecting the presence of a target nucleic acid in a sample can be used in a rapid lab tests for detection of a target nucleic acid of interest (e.g., target nucleic acids from influenza, coronavirus, or other pathogens, or target nucleic acids corresponding to a gene of interest). In particular, provided herein are devices, systems, fluidic devices, and kits, wherein the rapid lab tests can be performed in a single system. The target nucleic acid may be a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease in the sample. The target nucleic acid may be a portion of an RNA or DNA from any organism in the sample. In some embodiments, programmable nucleases disclosed herein are activated to initiate trans cleavage activity of an RNA reporter by RNA or DNA. A programmable nuclease as disclosed herein is, in some cases, binds to a target RNA to initiate trans cleavage of an RNA reporter, and this programmable nuclease can be referred to as an RNA-activated programmable RNA nuclease. In some instances, a programmable nuclease as disclosed herein binds to a target DNA to initiate trans cleavage of an RNA reporter, and this programmable nuclease can be referred to as a DNA-activated programmable RNA nuclease. In some cases, a programmable nuclease as described herein is capable of being activated by a target RNA or a target DNA. For example, a Cas13 protein, such as Cas13a, disclosed herein is activated by a target RNA nucleic acid or a target DNA nucleic acid to transcollaterally cleave RNA reporter molecules. In some embodiments, the Cas13 binds to a target ssDNA which initiates trans cleavage of RNA reporters. The detection of the target nucleic acid in the sample may indicate the presence of the disease in the sample and may provide information for taking action to reduce the transmission of the disease to individuals in the disease-affected environment or near the disease-carrying individual. The detection of the target nucleic acid in the sample may indicate the presence of a disease mutation, such as a single nucleotide polymorphism (SNP) that provide antibiotic resistance to a disease-causing bacteria. The detection of the target nucleic acid is facilitated by a programmable nuclease. The programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic, in which the activated programmable nuclease can cleave the target nucleic acid and can have trans cleavage activity, which can also be referred to as “collateral” or “transcollateral” cleavage. Trans cleavage activity can be non-specific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety. Once the detector nucleic acid is cleaved by the activated programmable nuclease, the detection moiety is released from the detector nucleic acid and generates a detectable signal that is immobilized to on a support medium. Often the detection moiety is at least one of a fluorophore, a dye, a polypeptide, or a nucleic acid. Sometimes the detection moiety binds to a capture molecule on the support medium to be immobilized. The detectable signal can be visualized on the support medium to assess the presence or level of the target nucleic acid associated with an ailment, such as a disease. The programmable nuclease can be a CRISPR-Cas (clustered regularly interspaced short palindromic repeats—CRISPR associated) nucleoprotein complex with trans cleavage activity, which can be activated by binding of a guide nucleic acid with a target nucleic acid.
In one aspect, described herein, is a system for detecting a target nucleic acid. The system may comprise a support medium; a guide nucleic acid targeting a target sequence; a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target sequence; and a single stranded detector nucleic acid comprising a detection moiety, wherein the detector nucleic acid is capable of being cleaved by the activated nuclease, thereby generating a first detectable signal.
In another aspect, described herein is a system for detecting a target nucleic acid, the system comprising a reagent chamber and a support medium for detection of the first detectable signal. The reagent chamber comprises a guide nucleic acid targeting a target sequence; a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target sequence; and a single stranded detector nucleic acid comprising a detection moiety, wherein the detector nucleic acid is capable of being cleaved by the activated nuclease, thereby generating a first detectable signal.
Further described herein is a method of detecting a target nucleic acid in a sample comprising contacting the sample with a guide nucleic acid targeting a target sequence, a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target sequence, a single stranded detector nucleic acid comprising a detection moiety, wherein the detector nucleic acid is capable of being cleaved by the activated nuclease, thereby generating a first detectable signal, and presenting the first detectable signal using a support medium.
Also described herein are various designs of assays for CRISPR-Cas diagnostics for detecting target nucleic acids (e.g., from influenza, coronavirus, or genes associated with a disease state). The design and format of the lateral flow assays disclosed herein can include new Cas reporter molecules, which can be tethered to the surface of the assay in a reaction chamber that is upstream of the lateral flow strip itself. The assay designs disclosed herein provide significant advantages as they minimize the chances of false positives, and thus can have improved sensitivity and specificity for a target nucleic acid.
Also described herein is a kit for detecting a target nucleic acid (e.g., from influenza, coronavirus, or genes associated with a disease state). The kit may comprise a support medium; a guide nucleic acid targeting a target sequence; a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target sequence; and a single stranded detector nucleic acid comprising a detection moiety, wherein the detector nucleic acid is capable of being cleaved by the activated nuclease, thereby generating a first detectable signal.
A biological sample from an individual or an environmental sample can be tested to determine whether the individual has a communicable disease. The biological sample can be tested to detect the presence or absence of at least one target nucleic acid from virus (e.g., an influenza virus, a coronavirus, or a respiratory syncytial virus). The biological sample can be tested to detect the presence or absence of at least one target nucleic acid from bacterium. The at least one target nucleic acid from a pathogen responsible for the disease that is detected can also indicate that the pathogen is wild-type or comprises a mutation that confers resistance to treatment, such as antibiotic treatment. In some embodiments, a biological sample from an individual or an environmental sample can be tested to determine whether the individual has a gene or gene mutation associated with a disease state. A sample from an individual or from an environment is applied to the reagents described herein. The reaction between the sample and the reagents may be performed in the reagent chamber provided in the kit or on a support medium provided in the kit. If the target nucleic acid is present in the sample, the target nucleic acid binds to the guide nucleic acid to activate the programmable nuclease. The activated programmable nuclease cleaves the detector nucleic acid and generates a detectable signal that can be visualized on the support medium. If the target nucleic acid is absent in the sample or below the threshold of detection, the guide nucleic acid remains unbound, the programmable nuclease remains inactivated, and the detector nucleic acid remains uncleaved. After the sample and the reagents are contacted for a predetermined time, the reacted sample is placed on a sample pad of a support medium. The sample can be placed on to the sample pad by dipping the support medium into the reagent chamber, applying the reacted sample to the sample pad, or allowing the sample to transport if the reagent was initially placed on the support medium. As the reacted sample and reagents move along the support medium to a detection region and after a predetermined amount of time after applying the reacted sample, a positive control marker can be visualized in the detection region. If the sample is positive for the target nucleic acid, a test marker for the detectable signal can also be visualized. The results in the detection region can be visualized by eye or using a mobile device. In some instances, an individual can open a mobile application for reading of the test results on a mobile device having a camera and take an image of the support medium, including the detection region, barcode, reference color scale, and fiduciary markers on the housing, using the camera of the mobile device and the graphic user interface (GUI) of the mobile application. The mobile application can identify the test, visualize the detection region in the image, and analyze to determine the presence or absence or the level of the target nucleic acid responsible for the disease. The mobile application can present the results of the test to the individual, store the test results in the mobile application, or communicate with a remote device and transfer the data of the test results.
Such devices, systems, fluidic devices, kits, and methods described herein may allow for detection of target nucleic acid, and in turn the viral infection (e.g., influenza viral infection, a coronavirus, or a respiratory syncytial virus), bacterial infection, or disease state associated with the target nucleic acid, in remote regions or low resource settings without specialized equipment. Also, such devices, systems, fluidic devices, kits, and methods described herein may allow for detection of target nucleic acid, and in turn the pathogen and disease associated with the target nucleic acid, in healthcare clinics or doctor offices without specialized equipment. In some cases, this provides a point of care testing for users to easily test for a disease or infection at home or quickly in an office of a healthcare provider. Assays that deliver results in under an hour, for example, in 15 to 60 minutes, are particularly desirable for at home testing for many reasons. Antivirals can be most effective when administered within the first 48 hours and improve antibiotic stewardship. Thus, the systems and assays disclosed herein, which are capable of delivering results in under an hour can will allow for the delivery of anti-viral therapy at an optimal time. Additionally, the systems and assays provided herein, which are capable of delivering quick diagnoses and results, can help keep or send a patient at home, improve comprehensive disease surveillance, and prevent the spread of an infection. In other cases, this provides a test, which can be used in a lab to detect a nucleic acid of interest in a sample from a subject. In particular, provided herein are devices, systems, fluidic devices, and kits, wherein the rapid lab tests can be performed in a single system. In some cases, this may be valuable in detecting diseases in a developing country and as a global healthcare tool to detect the spread of a disease or efficacy of a treatment or provide early detection of a viral infection, such as influenza.
Some methods as described herein use an editing technique, such as a technique using an editing enzyme or a programmable nuclease and guide nucleic acid, to detect a target nucleic acid. An editing enzyme or a programmable nuclease in the editing technique can be activated by a target nucleic acid, after which the activated editing enzyme or activated programmable nuclease can cleave nearby single-stranded nucleic acids, such detector nucleic acids with a detection moiety. A target nucleic acid (e.g., a target nucleic acid from a virus, such as influenza) can be amplified by isothermal amplification and then an editing technique can be used to detect the marker. In some instances, the editing technique can comprise an editing enzyme or programmable nuclease that, when activated, cleaves nearby RNA or DNA as the readout of the detection. The methods as described herein in some instances comprise obtaining a cell-free DNA sample, amplifying DNA from the sample, using an editing technique to cleave detector nucleic acids, and reading the output of the editing technique. In other instances, the method comprises obtaining a fluid sample from a patient, and without amplifying a nucleic acid of the fluid sample, using an editing technique to cleave detector nucleic acids, and detecting the nucleic acid. The method can also comprise using single-stranded detector DNA, cleaving the single-stranded detector DNA using an activated editing enzyme, wherein the editing enzyme cleaves at least 50% of a population of single-stranded detector DNA as measured by a change in color. A number of samples, guide nucleic acids, programmable nucleases or editing enzymes, support mediums, target nucleic acids, single-stranded detector nucleic acids, and reagents are consistent with the devices, systems, fluidic devices, kits, and methods disclosed herein.
Also disclosed herein are detector nucleic acids and methods detecting a target nucleic using the detector nucleic acids. Often, the detector nucleic acid is a protein-nucleic acid. For example, a method of assaying for a target nucleic acid in a sample comprises contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; and assaying for a signal indicating cleavage of at least some protein-nucleic acids of a population of protein-nucleic acids, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. Often, the protein-nucleic acid is an enzyme-nucleic acid or an enzyme substrate-nucleic acid. Sometimes, the protein-nucleic acid is attached to a solid support. The nucleic acid can be DNA, RNA, or a DNA/RNA hybrid. The methods described herein use a programmable nuclease, such as the CRISPR/Cas system, to detect a target nucleic acid. A method of assaying for a target nucleic acid in a sample, for example, comprises: a) contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; b) contacting the complex to a substrate; c) contacting the substrate to a reagent that differentially reacts with a cleaved substrate; and d) assaying for a signal indicating cleavage of the substrate, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. Often, the substrate is an enzyme-nucleic acid. Sometimes, the substrate is an enzyme substrate-nucleic acid.
Cleavage of the protein-nucleic acid produces a signal. For example, cleavage of the protein-nucleic acid produces a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo-electric signal. Various devices can be used to detect these different types signals, which indicate whether a target nucleic acid is present in the sample.
A number of samples are consistent with the devices, systems, fluidic devices, kits, and methods disclosed herein. These samples are, for example, consistent with fluidic devices disclosed herein for detection of a target nucleic acid within the sample, wherein the fluidic device may comprise multiple pumps, valves, reservoirs, and chambers for sample preparation, amplification of a target nucleic acid within the sample, mixing with a programmable nuclease, and detection of a detectable signal arising from cleavage of detector nucleic acids by the programmable nuclease within the fluidic system itself. These samples can comprise a target nucleic acid for detection of an ailment, such as a disease, pathogen, or virus, such as influenza. Generally, a sample from an individual or an animal or an environmental sample can be obtained to test for presence of a disease, or any mutation of interest. A biological sample from the individual may be blood, serum, plasma, saliva, urine, mucosal sample, peritoneal sample, cerebrospinal fluid, gastric secretions, nasal secretions, sputum, pharyngeal exudates, urethral or vaginal secretions, an exudate, an effusion, or tissue. A tissue sample may be dissociated or liquefied prior to application to detection system of the present disclosure. Samples can comprise one or more target nucleic acids for detection of an ailment, such as a disease, cancer, or genetic disorder, or genetic information, such as for phenotyping, genotyping, or determining ancestry and are compatible with the reagents and support mediums as described herein. Generally, a sample can be taken from any place where a nucleic acid can be found. Samples can be taken from an individual/human, a non-human animal, or a crop, or an environmental sample can be obtained to test for presence of a disease, virus, pathogen, cancer, genetic disorder, or any mutation or pathogen of interest. A biological sample can be blood, serum, plasma, lung fluid, exhaled breath condensate, saliva, spit, urine, stool, feces, mucus, lymph fluid, peritoneal, cerebrospinal fluid, amniotic fluid, breast milk, gastric secretions, bodily discharges, secretions from ulcers, pus, nasal secretions, sputum, pharyngeal exudates, urethral secretions/mucus, vaginal secretions/mucus, anal secretion/mucus, semen, tears, an exudate, an effusion, tissue fluid, interstitial fluid (e.g., tumor interstitial fluid), cyst fluid, tissue, or, in some instances, a combination thereof. A sample can be an aspirate of a bodily fluid from an animal (e.g. human, animals, livestock, pet, etc.) or plant. A tissue sample can be from any tissue that may be infected or affected by a pathogen (e.g., a wart, lung tissue, skin tissue, and the like). A tissue sample (e.g., from animals, plants, or humans) can be dissociated or liquified prior to application to detection system of the present disclosure. A sample can be from a plant (e.g., a crop, a hydroponically grown crop or plant, and/or house plant). Plant samples can include extracellular fluid, from tissue (e.g., root, leaves, stem, trunk etc.). A sample can be taken from the environment immediately surrounding a plant, such as hydroponic fluid/water, or soil. A sample from an environment may be from soil, air, or water. In some instances, the environmental sample is taken as a swab from a surface of interest or taken directly from the surface of interest. In some instances, the raw sample is applied to the detection system. In some instances, the sample is diluted with a buffer or a fluid or concentrated prior to application to the detection system or be applied neat to the detection system. Sometimes, the sample is contained in no more 20 uL. The sample, in some cases, is contained in no more than 1, 5, 10, 15, 20, 25, 30, 35 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500 uL, or any of value from 1 uL to 500 uL. Sometimes, the sample is contained in more than 500 uL.
In some instances, the sample is taken from single-cell eukaryotic organisms; a plant or a plant cell; an algal cell; a fungal cell; an animal cell, tissue, or organ; a cell, tissue, or organ from an invertebrate animal; a cell, tissue, fluid, or organ from a vertebrate animal such as fish, amphibian, reptile, bird, and mammal; a cell, tissue, fluid, or organ from a mammal such as a human, a non-human primate, an ungulate, a feline, a bovine, an ovine, and a caprine. In some instances, the sample is taken from nematodes, protozoans, helminths, or malarial parasites. In some cases, the sample comprises nucleic acids from a cell lysate from a eukaryotic cell, a mammalian cell, a human cell, a prokaryotic cell, or a plant cell. In some cases, the sample comprises nucleic acids expressed from a cell.
The sample used for disease testing may comprise at least one target sequence that can bind to a guide nucleic acid of the reagents described herein. In some cases, the target sequence is a portion of a nucleic acid. A portion of a nucleic acid can be from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA. A portion of a nucleic acid can be from 5 to 100, 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 nucleotides in length. A portion of a nucleic acid can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides in length. The target sequence can be reverse complementary to a guide nucleic acid.
In some cases, the target sequence is a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease in the sample. The target sequence, in some cases, is a portion of a nucleic acid from a sexually transmitted infection or a contagious disease, in the sample. The target sequence, in some cases, is a portion of a nucleic acid from an upper respiratory tract infection, a lower respiratory tract infection, or a contagious disease, in the sample. The target sequence, in some cases, is a portion of a nucleic acid from a hospital acquired infection or a contagious disease, in the sample. The target sequence, in some cases, is an ssRNA. These target sequences may be from a disease, and the disease may include but is not limited to influenza virus including influenza A virus (IAV) or influenza B virus (IBV), rhinovirus, cold viruses, a respiratory virus, an upper respiratory virus, a lower respiratory virus, or respiratory syncytial virus. Pathogens include viruses, fungi, helminths, protozoa, and parasites. Pathogenic viruses include but are not limited to influenza virus and the like. Pathogens include, e.g., Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria meningitidis, Pneumococcus, Hemophilus influenzae B, influenza virus, respiratory syncytial virus (RSV), M. pneumoniae, Streptococcus intermdius, Streptococcus pneumoniae, and Streptococcus pyogenes. Often the target nucleic acid comprises a sequence from a virus or a bacterium or other agents responsible for a disease that can be found in the sample. Pathogenic viruses include but are not limited to influenza virus; RSV; coronavirus, an ssRNA virus, a respiratory virus, an upper respiratory virus, a lower respiratory virus, or a rhinovirus. Pathogens include, e.g., Mycobacterium tuberculosis, Streptococcus agalactiae, Legionella pneumophila, Streptococcus pyogenes, Hemophilus influenzae B influenza virus, respiratory syncytial virus (RSV), or Mycobacterium tuberculosis
In some cases, the target sequence is a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease in the sample. The target sequence, in some cases, is a portion of a nucleic acid from a sexually transmitted infection or a contagious disease, in the sample. The target sequence, in some cases, is a portion of a nucleic acid from an upper respiratory tract infection, a lower respiratory tract infection, or a contagious disease, in the sample. The target sequence, in some cases, is a portion of a nucleic acid from a hospital acquired infection or a contagious disease, in the sample. The target sequence, in some cases, is a portion of a nucleic acid from sepsis, in the sample. These diseases can include but are not limited to respiratory viruses (e.g., COVID-19, SARS, MERS, influenza and the like) human immunodeficiency virus (HIV), human papillomavirus (HPV), chlamydia, gonorrhea, syphilis, trichomoniasis, sexually transmitted infection, malaria, Dengue fever, Ebola, chikungunya, and leishmaniasis. Pathogens include viruses, fungi, helminths, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites. Helminths include roundworms, heartworms, and phytophagous nematodes, flukes, Acanthocephala, and tapeworms. Protozoan infections include infections from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis. Examples of pathogens such as parasitic/protozoan pathogens include, but are not limited to: Plasmodium falciparum, P. vivax, Trypanosoma cruzi and Toxoplasma gondii. Fungal pathogens include, but are not limited to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitides, Chlamydia trachomatis, and Candida albicans. Pathogenic viruses include but are not limited to: respiratory viruses (e.g., adenoviruses, parainfluenza viruses, severe acute respiratory syndrome (SARS), coronavirus, MERS), gastrointestinal viruses (e.g., noroviruses, rotaviruses, some adenoviruses, astroviruses), exanthematous viruses (e.g. the virus that causes measles, the virus that causes rubella, the virus that causes chickenpox/shingles, the virus that causes roseola, the virus that causes smallpox, the virus that causes fifth disease, chikungunya virus infection); hepatic viral diseases (e.g., hepatitis A, B, C, D, E); cutaneous viral diseases (e.g. warts (including genital, anal), herpes (including oral, genital, anal), molluscum contagiosum); hemmorhagic viral diseases (e.g. Ebola, Lassa fever, dengue fever, yellow fever, Marburg hemorrhagic fever, Crimean-Congo hemorrhagic fever); neurologic viruses (e.g., polio, viral meningitis, viral encephalitis, rabies), sexually transmitted viruses (e.g., HIV, HPV, and the like), immunodeficiency virus (e.g., HIV); influenza virus; dengue; West Nile virus; herpes virus; yellow fever virus; Hepatitis Virus C; Hepatitis Virus A; Hepatitis Virus B; papillomavirus; and the like. Pathogens include, e.g., HIV virus, Mycobacterium tuberculosis, Klebsiella pneumoniae, Acinetobacter baumannii, Burkholderia cepacia, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, human serum parvo-like virus, respiratory syncytial virus (RSV), M. genitalium, T. Vaginalis, varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus, feline leukemia virus, Reovirus, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, West Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocerca volvulus, Leishmania tropica, Mycobacterium tuberculosis, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium, M. pneumoniae, Enterobacter cloacae, Kiebsiella aerogenes, Proteus vulgaris, Serratia macesens, Enterococcus faecalis, Enterococcus faecium, Streptococcus intermdius, Streptococcus pneumoniae, and Streptococcus pyogenes. Often the target nucleic acid comprises a sequence from a virus or a bacterium or other agents responsible for a disease that can be found in the sample. In some cases, the target nucleic acid is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus in at least one of: human immunodeficiency virus (HIV), human papillomavirus (HPV), chlamydia, gonorrhea, syphilis, trichomoniasis, sexually transmitted infection, malaria, Dengue fever, Ebola, chikungunya, and leishmaniasis. Pathogens include viruses, fungi, helminths, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites. Helminths include roundworms, heartworms, and phytophagous nematodes, flukes, Acanthocephala, and tapeworms. Protozoan infections include infections from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis. Examples of pathogens such as parasitic/protozoan pathogens include, but are not limited to: Plasmodium falciparum, P. vivax, Trypanosoma cruzi and Toxoplasma gondii. Fungal pathogens include, but are not limited to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitides, Chlamydia trachomatis, and Candida albicans. Pathogenic viruses include but are not limited to immunodeficiency virus (e.g., HIV); influenza virus; dengue; West Nile virus; herpes virus; yellow fever virus; Hepatitis Virus C; Hepatitis Virus A; Hepatitis Virus B; papillomavirus; and the like. Pathogens include, e.g., HIV virus, Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, human serum parvo-like virus, respiratory syncytial virus (RSV), M. genitalium, T. vaginalis, varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus, feline leukemia virus, Reovirus, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, West Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocerca volvulus, Leishmania tropica, Mycobacterium tuberculosis, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium and M. pneumoniae. In some cases, the target sequence is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus of bacterium or other agents responsible for a disease in the sample comprising a mutation that confers resistance to a treatment, such as a single nucleotide mutation that confers resistance to antibiotic treatment.
The sample used for cancer testing or cancer risk testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene with a mutation associated with cancer, from a gene whose overexpression is associated with cancer, a tumor suppressor gene, an oncogene, a checkpoint inhibitor gene, a gene associated with cellular growth, a gene associated with cellular metabolism, or a gene associated with cell cycle. Sometimes, the target nucleic acid encodes for a cancer biomarker, such as a prostate cancer biomarker or non-small cell lung cancer. In some cases, the assay can be used to detect “hotspots” in target nucleic acids that can be predictive of cancer, such as lung cancer, cervical cancer, in some cases, the cancer can be a cancer that is caused by a virus. Some non-limiting examples of viruses that cause cancers in humans include Epstein-Barr virus (e.g., Burkitt's lymphoma, Hodgkin's Disease, and nasopharyngeal carcinoma); papillomavirus (e.g., cervical carcinoma, anal carcinoma, oropharyngeal carcinoma, penile carcinoma); hepatitis B and C viruses (e.g., hepatocellular carcinoma); human adult T-cell leukemia virus type 1 (HTLV-1) (e.g., T-cell leukemia); and Merkel cell polyomavirus (e.g., Merkel cell carcinoma). One skilled in the art will recognize that viruses can cause or contribute to other types of cancers. In some cases, the target nucleic acid is a portion of a nucleic acid that is associated with a blood fever. In some cases, the target nucleic acid segment is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a locus of at least one of: ALK, APC, ATM, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CASR, CDC73, CDH1, CDK4, CDKN1B, CDKN1C, CDKN2A, CEBPA, CHEK2, CTNNA1, DICER1, DIS3L2, EGFR, EPCAM, FH, FLCN, GATA2, GPC3, GREM1, HOXB13, HRAS, KIT, MAX, MEN1, MET, MITF, MLH1, MSH2, MSH3, MSH6, MUTYH, NBN, NF1, NF2, NTHL1, PALB2, PDGFRA, PHOX2B, PMS2, POLD1, POLE, POT1, PRKAR1A, PTCH1, PTEN, RAD50, RAD51C, RAD51D, RB1, RECQL4, RET, RUNX1, SDHA, SDHAF2, SDHB, SDHC, SDHD, SMAD4, SMARCA4, SMARCB1, SMARCE1, STK11, SUFU, TERC, TERT, TMEM127, TP53, TSC1, TSC2, VHL, WRN, and WT1.
The sample used for genetic disorder testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein. In some embodiments, the genetic disorder is hemophilia, sickle cell anemia, β-thalassemia, Duchene muscular dystrophy, severe combined immunodeficiency, or cystic fibrosis. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene with a mutation associated with a genetic disorder, from a gene whose overexpression is associated with a genetic disorder, from a gene associated with abnormal cellular growth resulting in a genetic disorder, or from a gene associated with abnormal cellular metabolism resulting in a genetic disorder. In some cases, the target nucleic acid segment is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a locus of at least one of: CFTR, FMR1, SMN1, ABCB11, ABCC8, ABCD1, ACAD9, ACADM, ACADVL, ACAT1, ACOX1, ACSF3, ADA, ADAMTS2, ADGRG1, AGA, AGL, AGPS, AGXT, AIRE, ALDH3A2, ALDOB, ALG6, ALMS1, ALPL, AMT, AQP2, ARG1, ARSA, ARSB, ASL, ASNS, ASPA, ASS1, ATM, ATP6V1B1, ATP7A, ATP7B, ATRX, BBS1, BBS10, BBS12, BBS2, BCKDHA, BCKDHB, BCS1L, BLM, BSND, CAPN3, CBS, CDH23, CEP290, CERKL, CHM, CHRNE, CIITA, CLN3, CLN5, CLN6, CLN8, CLRN1, CNGB3, COL27A1, COL4A3, COL4A4, COL4A5, COL7A1, CPS1, CPT1A, CPT2, CRB1, CTNS, CTSK, CYBA, CYBB, CYP11B1, CYP11B2, CYP17A1, CYP19A1, CYP27A1, DBT, DCLRE1C, DHCR7, DHDDS, DLD, DMD, DNAH5, DNAI1, DNAI2, DYSF, EDA, EIF2B5, EMD, ERCC6, ERCC8, ESCO2, ETFA, ETFDH, ETHE1, EVC, EVC2, EYS, F9, FAH, FAM161A, FANCA, FANCC, FANCG, FH, FKRP, FKTN, G6PC, GAA, GALC, GALK1, GALT, GAMT, GBA, GBE1, GCDH, GFM1, GJB1, GJB2, GLA, GLB1, GLDC, GLE1, GNE, GNPTAB, GNPTG, GNS, GRHPR, HADHA, HAX1, HBA1, HBA2, HBB, HEXA, HEXB, HGSNAT, HLCS, HMGCL, HOGA1, HPS1, HPS3, HSD17B4, HSD3B2, HYAL1, HYLS1, IDS, IDUA, IKBKAP, IL2RG, IVD, KCNJ11, LAMA2, LAMA3, LAMB3, LAMC2, LCA5, LDLR, LDLRAP1, LHX3, LIFR, LIPA, LOXHD1, LPL, LRPPRC, MAN2B1, MCOLN1, MED17, MESP2, MFSD8, MKS1, MLC1, MMAA, MMAB, MMACHC, MMADHC, MPI, MPL, MPV17, MTHFR, MTM1, MTRR, MTTP, MUT, MYO7A, NAGLU, NAGS, NBN, NDRG1, NDUFAF5, NDUFS6, NEB, NPC1, NPC2, NPHS1, NPHS2, NR2E3, NTRK1, OAT, OPA3, OTC, PAH, PC, PCCA, PCCB, PCDH15, PDHA1, PDHB, PEX1, PEX10, PEX12, PEX2, PEX6, PEX7, PFKM, PHGDH, PKHD1, PMM2, POMGNT1, PPT1, PROP1, PRPS1, PSAP, PTS, PUS1, PYGM, RAB23, RAG2, RAPSN, RARS2, RDH12, RMRP, RPE65, RPGRIP1L, RS1, RTEL1, SACS, SAMHD1, SEPSECS, SGCA, SGCB, SGCG, SGSH, SLC12A3, SLC12A6, SLC17A5, SLC22A5, SLC25A13, SLC25A15, SLC26A2, SLC26A4, SLC35A3, SLC37A4, SLC39A4, SLC4A11, SLC6A8, SLC7A7, SMARCAL1, SMPD1, STAR, SUMF1, TAT, TCIRG1, TECPR2, TFR2, TGM1, TH, TMEM216, TPP1, TRMU, TSFM, TTPA, TYMP, USH1C, USH2A, VPS13A, VPS13B, VPS45, VRK1, VSX2, WNT10A, XPA, XPC, and ZFYVE26.
In some embodiments, the target nucleic acid sequence comprises a nucleic acid sequence of a virus, a bacterium, or other pathogen responsible for a disease in a plant (e.g., a crop). Methods and compositions of the disclosure can be used to treat or detect a disease in a plant. For example, the methods of the disclosure can be used to target a viral nucleic acid sequence in a plant. A programmable nuclease of the disclosure can cleave the viral nucleic acid. In some embodiments, the target nucleic acid sequence comprises a nucleic acid sequence of a virus or a bacterium or other agents (e.g., any pathogen) responsible for a disease in the plant (e.g., a crop). In some embodiments, the target nucleic acid comprises DNA that is reverse transcribed from RNA using a reverse transcriptase prior to detection by a programmable nuclease using the compositions, systems, and methods disclosed herein. The target nucleic acid, in some cases, is a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease in the plant (e.g., a crop). In some cases, the target nucleic acid is a portion of a nucleic acid from a genomic locus, or any DNA amplicon, such as a reverse transcribed mRNA or a cDNA from a gene locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus in at a virus or a bacterium or other agents (e.g., any pathogen) responsible for a disease in the plant (e.g., a crop). A virus infecting the plant can be an RNA virus. A virus infecting the plant can be a DNA virus. Non-limiting examples of viruses that can be targeted with the disclosure include Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), Cauliflower mosaic virus (CaMV) (RT virus), Plum pox virus (PPV), Brome mosaic virus (BMV) and Potato virus X (PVX).
The plant can be a monocotyledonous plant. The plant can be a dicotyledonous plant. Non-limiting examples of orders of dicotyledonous plants include Magniolales, Illiciales, Laurales, Piperales, Aristochiales, Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales, Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales, Lecythidales, Violales, Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales, Myrtales, Cornales, Proteales, San tales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales, Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales, and Asterales.
Non-limiting examples of orders of monocotyledonous plants include Alismatales, Hydrocharitales, Najadales, Triuridales, Commelinales, Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales, Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales, Lilliales, and Orchid ales. A plant can belong to the order, for example, Gymnospermae, Pinales, Ginkgoales, Cycadales, Araucariales, Cupressales and Gnetales.
Non-limiting examples of plants include plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses, wheat, maize, rice, millet, barley, tomato, apple, pear, strawberry, orange, acacia, carrot, potato, sugar beets, yam, lettuce, spinach, sunflower, rape seed, Arabidopsis, alfalfa, amaranth, apple, apricot, artichoke, ash tree, asparagus, avocado, banana, barley, beans, beet, birch, beech, blackberry, blueberry, broccoli, Brussel's sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, cedar, a cereal, celery, chestnut, cherry, Chinese cabbage, citrus, clementine, clover, coffee, corn, cotton, cowpea, cucumber, cypress, eggplant, elm, endive, eucalyptus, fennel, figs, fir, geranium, grape, grapefruit, groundnuts, ground cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch, lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango, maple, melon, millet, mushroom, mustard, nuts, oak, oats, oil palm, okra, onion, orange, an ornamental plant or flower or tree, papaya, palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper, persimmon, pigeon pea, pine, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, safflower, sallow, soybean, spinach, spruce, squash, strawberry, sugar beet, sugarcane, sunflower, sweet potato, sweet corn, tangerine, tea, tobacco, tomato, trees, triticale, turf grasses, turnips, vine, walnut, watercress, watermelon, wheat, yams, yew, and zucchini. A plant can include algae.
The sample can be used for identifying a disease status. For example, a sample is any sample described herein, and is obtained from a subject for use in identifying a disease status of a subject. Sometimes, a method comprises obtaining a serum sample from a subject; and identifying a disease status of the subject.
In some instances, the target nucleic acid is a single stranded nucleic acid. Alternatively or in combination, the target nucleic acid is a double stranded nucleic acid and is prepared into single stranded nucleic acids before or upon contacting the reagents. The target nucleic acid may be a RNA, DNA, synthetic nucleic acids, or nucleic acids found in biological or environmental samples. The target nucleic acids include but are not limited to mRNA, rRNA, tRNA, non-coding RNA, long non-coding RNA, and microRNA (miRNA). In some cases, the target nucleic acid is mRNA. In some cases, the target nucleic acid is from a virus, a parasite, or a bacterium described herein. In some cases, the target nucleic acid is transcribed from a gene as described herein.
A number of target nucleic acids are consistent with the methods and compositions disclosed herein. Some methods described herein can detect a target nucleic acid present in the sample in various concentrations or amounts as a target nucleic acid population. In some cases, the sample has at least 2 target nucleic acids. In some cases, the sample has at least 3, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 target nucleic acids. In some cases, the method detects target nucleic acid present at least at one copy per 101 non-target nucleic acids, 102 non-target nucleic acids, 103 non-target nucleic acids, 104 non-target nucleic acids, 105 non-target nucleic acids, 106 non-target nucleic acids, 107 non-target nucleic acids, 108 non-target nucleic acids, 109 non-target nucleic acids, or 1010 non-target nucleic acids.
A number of target nucleic acid populations are consistent with the methods and compositions disclosed herein. Some methods described herein can detect two or more target nucleic acid populations present in the sample in various concentrations or amounts. In some cases, the sample has at least 2 target nucleic acid populations. In some cases, the sample has at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 target nucleic acid populations. In some cases, the method detects target nucleic acid populations that are present at least at one copy per 101 non-target nucleic acids, 102 non-target nucleic acids, 103 non-target nucleic acids, 104 non-target nucleic acids, 105 non-target nucleic acids, 106 non-target nucleic acids, 107 non-target nucleic acids, 108 non-target nucleic acids, 109 non-target nucleic acids, or 1010 non-target nucleic acids. The target nucleic acid populations can be present at different concentrations or amounts in the sample.
Any of the above disclosed samples are consistent with the systems, assays, and programmable nucleases disclosed herein and can be used as a companion diagnostic with any of the diseases disclosed herein (e.g., influenza A, influenza B, RSV), or can be used in reagent kits, point-of-care diagnostics, or over-the-counter diagnostics.
Reagents A number of reagents are consistent with the devices, systems, fluidic devices, kits, and methods disclosed herein. These reagents are, for example, consistent for use within various fluidic devices disclosed herein for detection of a target nucleic acid (e.g., influenza A or influenza B) within the sample, wherein the fluidic device may comprise multiple pumps, valves, reservoirs, and chambers for sample preparation, amplification of a target nucleic acid within the sample, mixing with a programmable nuclease, and detection of a detectable signal arising from cleavage of detector nucleic acids by the programmable nuclease within the fluidic system itself. These reagents are compatible with the samples, fluidic devices, and support mediums as described herein for detection of an ailment, such as a disease. The reagents described herein for detecting a disease, such as influenza or RSV, comprise a guide nucleic acid targeting the target nucleic acid segment indicative of the disease. The guide nucleic acid binds to the single stranded target nucleic acid comprising a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease as described herein. The guide nucleic acid can bind to the single stranded target nucleic acid comprising a portion of a nucleic acid from a bacterium or other agents responsible for a disease as described herein and further comprising a mutation, such as a single nucleotide polymorphism (SNP), that can confer resistance to a treatment, such as antibiotic treatment. The guide nucleic acid binds to the single stranded target nucleic acid comprising a portion of a nucleic acid from an influenza virus, such as influenza A or influenza B. The guide nucleic acid is complementary to the target nucleic acid. Often the guide nucleic acid binds specifically to the target nucleic acid. The target nucleic acid may be a RNA, DNA, or synthetic nucleic acids.
Disclosed herein are methods of assaying for a target nucleic acid as described herein. For example, a method of assaying for a target nucleic acid in a sample comprises contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; and assaying for a signal indicating cleavage of at least some protein-nucleic acids of a population of protein-nucleic acids, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. As another example, a method of assaying for a target nucleic acid in a sample, for example, comprises: a) contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; b) contacting the complex to a substrate; c) contacting the substrate to a reagent that differentially reacts with a cleaved substrate; and d) assaying for a signal indicating cleavage of the substrate, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. Often, the substrate is an enzyme-nucleic acid. Sometimes, the substrate is an enzyme substrate-nucleic acid.
A programmable nuclease can comprise a programmable nuclease capable of being activated when complexed with a guide nucleic acid and target nucleic acid. The programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic acid, in which the activated programmable nuclease can cleave the target nucleic acid and can have trans cleavage activity. Trans cleavage activity can be non-specific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety. Once the detector nucleic acid is cleaved by the activated programmable nuclease, the detection moiety can be released from the detector nucleic acid and can generate a signal. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorometric, etc.), or piezo-electric signal. Often, the signal is present prior to detector nucleic acid cleavage and changes upon detector nucleic acid cleavage. Sometimes, the signal is absent prior to detector nucleic acid cleavage and is present upon detector nucleic acid cleavage. The detectable signal can be immobilized on a support medium for detection. The programmable nuclease can be a CRISPR-Cas (clustered regularly interspaced short palindromic repeats—CRISPR associated) nucleoprotein complex with trans cleavage activity, which can be activated by binding of a guide nucleic acid with a target nucleic acid. The CRISPR-Cas nucleoprotein complex can comprise a Cas protein (also referred to as a Cas nuclease) complexed with a guide nucleic acid, which can also be referred to as CRISPR enzyme. A guide nucleic acid can be a CRISPR RNA (crRNA). Sometimes, a guide nucleic acid comprises a crRNA and a trans-activating crRNA (tracrRNA).
The CRISPR/Cas system used to detect a modified target nucleic acids can comprise CRISPR RNAs (crRNAs), trans-activating crRNAs (tracrRNAs), Cas proteins, and detector nucleic acids.
A guide nucleic acid can comprise a sequence that is reverse complementary to the sequence of a target nucleic acid. A guide nucleic acid can be a crRNA. Sometimes, a guide nucleic acid comprises a crRNA and tracrRNA. The guide nucleic acid can bind specifically to the target nucleic acid. In some cases, the guide nucleic acid is not naturally occurring and made by artificial combination of otherwise separate segments of sequence. Often, the artificial combination is performed by chemical synthesis, by genetic engineering techniques, or by the artificial manipulation of isolated segments of nucleic acids. The target nucleic acid can be designed and made to provide desired functions. In some cases, the targeting region of a guide nucleic acid is 20 nucleotides in length. The targeting region of the guide nucleic acid may have a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some instances, the targeting region of the guide nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, the targeting region of a guide nucleic acid has a length from exactly or about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable or bind specifically. The guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a modification variable region in the target nucleic acid. The guide nucleic acid, in some cases, has a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a modification variable region in the target nucleic acid. The guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid. The guide nucleic acid, in some cases, has a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid.
The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid sequence of a strain of an infection or genomic locus of interest. The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid sequence of a strain of influenza A or influenza B. Often, guide nucleic acids that are tiled against the nucleic acid of a strain of an infection or genomic locus of interest can be pooled for use in a method described herein. Often, these guide nucleic acids are pooled for detecting a target nucleic acid in a single assay. The pooling of guide nucleic acids that are tiled against a single target nucleic acid can enhance the detection of the target nucleic using the methods described herein. The pooling of guide nucleic acids that are tiled against a single target nucleic acid can ensure broad coverage of the target nucleic acid within a single reaction using the methods described herein. The tiling, for example, is sequential along the target nucleic acid. Sometimes, the tiling is overlapping along the target nucleic acid. In some instances, the tiling comprises gaps between the tiled guide nucleic acids along the target nucleic acid. In some instances the tiling of the guide nucleic acids is non-sequential. Often, a method for detecting a target nucleic acid comprises contacting a target nucleic acid to a pool of guide nucleic acids and a programmable nuclease, wherein a guide nucleic acid of the pool of guide nucleic acids has a sequence selected from a group of tiled guide nucleic acid that correspond to nucleic acids of a target nucleic acid; and assaying for a signal produce by cleavage of at least some detector nucleic acids of a population of detector nucleic acids. Pooling of guide nucleic acids can ensure broad spectrum identification, or broad coverage, of a target species within a single reaction. This can be particularly helpful in diseases or indications, like sepsis, that may be caused by multiple organisms.
Described herein are reagents comprising a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment. A programmable nuclease can be capable of being activated when complexed with a guide nucleic acid and the target sequence. The programmable nuclease can be activated upon binding of the guide nucleic acid to its target nucleic acid and degrades non-specifically nucleic acid in its environment. The programmable nuclease has trans cleavage activity once activated. A programmable nuclease can be a Cas protein (also referred to, interchangeably, as a Cas nuclease). A crRNA and Cas protein can form a CRISPR enzyme.
“Percent identity” and “% identity” can refer to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X % identical to SEQ ID NO: Y” can refer to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X % of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y. Generally, computer programs can be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci. 1988 March; 4(1):11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci USA. 1988 April; 85(8):2444-8; Pearson, Methods Enzymol. 1990; 183:63-98) and gapped BLAST (Altschul et al., Nucleic Acids Res. 1997 Sep. 1; 25(17):3389-40), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic Acids Res. 1984 Jan. 11; 12(1 Pt 1):387-95).
Several programmable nucleases are consistent with the methods and devices of the present disclosure. For example, CRISPR/Cas enzymes are programmable nucleases used in the methods and systems disclosed herein. CRISPR/Cas enzymes can include any of the known Classes and Types of CRISPR/Cas enzymes. Programmable nucleases disclosed herein include Class 1 CRISPR/Cas enzymes, such as the Type I, Type IV, or Type III CRISPR/Cas enzymes. Programmable nucleases disclosed herein also include the Class 2 CRISPR/Cas enzymes, such as the Type II, Type V, and Type VI CRISPR/Cas enzymes. Preferable programmable nucleases included in the several devices disclosed herein (e.g., a microfluidic device such as a pneumatic valve device or a sliding valve device or a lateral flow assay) and methods of use thereof include a Type V or Type VI CRISPR/Cas enzyme.
In some embodiments, the Type V CRISPR/Cas enzyme is a programmable Cas12 nuclease. Type V CRISPR/Cas enzymes (e.g., Cas12 or Cas14) lack an HNH domain. A Cas12 nuclease of the present disclosure cleaves a nucleic acids via a single catalytic RuvC domain. The RuvC domain is within a nuclease, or “NUC” lobe of the protein, and the Cas12 nucleases further comprise a recognition, or “REC” lobe. The REC and NUC lobes are connected by a bridge helix and the Cas12 proteins additionally include two domains for PAM recognition termed the PAM interacting (PI) domain and the wedge (WED) domain. (Murugan et al., Mol Cell. 2017 Oct. 5; 68(1): 15-25). A programmable Cas12 nuclease can be a Cas12a (also referred to as Cpf1) protein, a Cas12b protein, Cas12c protein, Cas12d protein, or a Cas12e protein. In some cases, a suitable Cas12 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to any one of SEQ ID NO: 27-SEQ ID NO: 37.
Lachnospiraceae
bacterium
Acidaminococcus
Francisella
novicida
Porphyromonas
macacae
Moraxella
bovoculi
Moraxella
bovoculi
Moraxella
bovoculi
Thiomicrospira
Butyrivibrio
Alternatively, the Type V CRISPR/Cas enzyme is a programmable Cas14 nuclease. A Cas14 protein of the present disclosure includes 3 partial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains) that are not contiguous with respect to the primary amino acid sequence of the Cas14 protein, but form a RuvC domain once the protein is produced and folds. A naturally occurring Cas14 protein functions as an endonuclease that catalyzes cleavage at a specific sequence in a target nucleic acid. A programmable Cas14 nuclease can be a Cas14a protein, a Cas14b protein, a Cas14c protein, a Cas14d protein, a Cas14e protein, a Cas 14f protein, a Cas14g protein, a Cas14h protein, or a Cas14u protein. In some cases, a suitable Cas14 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to any one of SEQ ID NO: 38-SEQ ID NO: 129.
In some embodiments, the Type V CRISPR/Cas enzyme is a CasΦ nuclease. A CasΦ polypeptide can function as an endonuclease that catalyzes cleavage at a specific sequence in a target nucleic acid. A programmable CasΦ nuclease of the present disclosure may have a single active site in a RuvC domain that is capable of catalyzing pre-crRNA processing and nicking or cleaving of nucleic acids. This compact catalytic site may render the programmable CasΦ nuclease especially advantageous for genome engineering and new functionalities for genome manipulation.
TABLE 3 provides amino acid sequences of illustrative Case polypeptides that can be used in compositions and methods of the disclosure.
AGQAKKKKEF
In some embodiments, any of the programmable CasΦ nuclease of the present disclosure (e.g., any one of SEQ ID NO: 274-SEQ ID NO: 321 or fragments or variants thereof) may include a nuclear localization signal (NLS). In some cases, said NLS may have a sequence of KRPAATKKAGQAKKKKEF (SEQ ID NO: 322).
A CasΦ polypeptide or a variant thereof can comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 274-SEQ ID NO: 321.
In some embodiments, the Type VI CRISPR/Cas enzyme is a programmable Cas13 nuclease. The general architecture of a Cas13 protein includes an N-terminal domain and two HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domains separated by two helical domains (Liu et al., Cell 2017 Jan. 12; 168(1-2):121-134.e12). The HEPN domains each comprise aR-X4-H motif. Shared features across Cas13 proteins include that upon binding of the crRNA of the guide nucleic acid to a target nucleic acid, the protein undergoes a conformational change to bring together the HEPN domains and form a catalytically active RNase. (Tambe et al., Cell Rep. 2018 Jul. 24; 24(4): 1025-1036.). Thus, two activatable HEPN domains are characteristic of a programmable Cas13 nuclease of the present disclosure. However, programmable Cas13 nucleases also consistent with the present disclosure include Cas13 nucleases comprising mutations in the HEPN domain that enhance the Cas13 proteins cleavage efficiency or mutations that catalytically inactivate the HEPN domains. Programmable Cas13 nucleases consistent with the present disclosure also Cas13 nucleases comprising catalytic
A programmable Cas13 nuclease can be a Cas13a protein (also referred to as “c2c2”), a Cas13b protein, a Cas13c protein, a Cas13d protein, or a Cas13e protein. Example C2c2 proteins are set forth as SEQ ID NO: 130-SEQ ID NO: 137. In some cases, a subject C2c2 protein includes an amino acid sequence having 80% or more (e.g., 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100%) amino acid sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 130-SEQ ID NO: 137. In some cases, a suitable C2c2 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Listeria seeligeri C2c2 amino acid sequence set forth in SEQ ID NO: 130. In some cases, a suitable C2c2 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Leptotrichia buccalis C2c2 amino acid sequence set forth in SEQ ID NO: 131. In some cases, a suitable C2c2 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Rhodobacter capsulatus C2c2 amino acid sequence set forth in SEQ ID NO: 133. In some cases, a suitable C2c2 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Carnobacterium gallinarum C2c2 amino acid sequence set forth in SEQ ID NO: 134. In some cases, a suitable C2c2 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Herbinix hemicellulosilytica C2c2 amino acid sequence set forth in SEQ ID NO: 135. In some cases, the C2c2 protein includes an amino acid sequence having 80% or more amino acid sequence identity with the Leptotrichia buccalis (Lbu) C2c2 amino acid sequence set forth in SEQ ID NO: 131. In some cases, the C2c2 protein is a Leptotrichia buccalis (Lbu) C2c2 protein (e.g., see SEQ ID NO: 131). In some cases, the C2c2 protein includes the amino acid sequence set forth in any one of SEQ ID NOs: 130-131 and SEQ ID NOs: 133-137. In some cases, a C2c2 protein used in a method of the present disclosure is not a Leptotrichia shahii (Lsh) C2c2 protein. In some cases, a C2c2 protein used in a method of the present disclosure is not a C2c2 polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Lsh C2c2 polypeptide set forth in SEQ ID NO: 132. Other Cas13 protein sequences are set forth in SEQ ID NO: 130-SEQ ID NO: 147.
Listeria
seeligeri C2c2
Leptotrichia
buccalis (Lbu)
Leptotrichia
shahii (Lsh)
Rhodobacter
capsulatus
Carnobacterium
gallinarum
Herbinix
hemi-
cellulosilytica
Paludibacter
propionicigenes
Leptotrichia
wadei (Lwa)
Bergeyella
zoohelcum
Prevotella
intermedia
Prevotella
buccae Cas13b
Porphyromonas
gingivalis
Bacteroides
pyogenes
The programmable nuclease can be Cas13. Sometimes the Cas13 can be Cas13a, Cas13b, Cas13c, Cas13d, or Cas13e. In some cases, the programmable nuclease can be Mad7 or Mad2. In some cases, the programmable nuclease can be Cas12. Sometimes the Cas12 can be Cas12a, Cas12b, Cas12c, Cas12d, or Cas12e. In some cases, the programmable nuclease can be Csm1, Cas9, C2c4, C2c8, C2c5, C2c10, C2c9, or CasZ. Sometimes, the Csm1 can also be also called smCms1, miCms1, obCms1, or suCms1. Sometimes Cas13a can also be also called C2c2. Sometimes CasZ can also be called Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, or Cas14h. Sometimes, the programmable nuclease can be a type V CRISPR-Cas system. In some cases, the programmable nuclease can be a type VI CRISPR-Cas system. Sometimes the programmable nuclease can be a type III CRISPR-Cas system. In some cases, the programmable nuclease can be from at least one of Leptotrichia shahii (Lsh), Listeria seeligeri (Lse), Leptotrichia buccalis (Lbu), Leptotrichia wadeu (Lwa), Rhodobacter capsulatus (Rca), Herbinix hemicellulosilytica (Hhe), Paludibacter propionicigenes (Ppr), Lachnospiraceae bacterium (Lba), [Eubacterium] rectale (Ere), Listeria newyorkensis (Lny), Clostridium aminophilum (Cam), Prevotella sp. (Psm), Capnocytophaga canimorsus (Cca, Lachnospiraceae bacterium (Lba), Bergeyella zoohelcum (Bzo), Prevotella intermedia (Pin), Prevotella buccae (Pbu), Alistipes sp. (Asp), Riemerella anatipestifer (Ran), Prevotella aurantiaca (Pau), Prevotella saccharolytica (Psa), Prevotella intermedia (Pint), Capnocytophaga canimorsus (Cca), Porphyromonas gulae (Pgu), Prevotella sp. (Psp), Porphyromonas gingivalis (Pig), Prevotella intermedia (Pin3), Enterococcus italicus (Ei), Lactobacillus salivarius (Ls), or Therms thermophilus (Tt). Sometimes the Cas13 is at least one of LbuCas13a, LwaCas13a, LbaCas13a, HheCas13a, PprCas13a, EreCas13a, CamCas13a, or LshCas13a. The trans cleavage activity of the CRISPR enzyme can be activated when the crRNA is complexed with the target nucleic acid. The trans cleavage activity of the CRISPR enzyme can be activated when the guide nucleic acid comprising a tracrRNA and crRNA are complexed with the target nucleic acid. The target nucleic acid can be RNA or DNA.
In some embodiments, a programmable nuclease as disclosed herein is an RNA-activated programmable RNA nuclease. In some embodiments, a programmable nuclease as disclosed herein is a DNA-activated programmable RNA nuclease. In some embodiments, a programmable nuclease is capable of being activated by a target RNA to initiate trans cleavage of an RNA reporter and is capable of being activated by a target DNA to initiate trans cleavage of an RNA reporter, such as a Type VI CRISPR/Cas enzyme (e.g., Cas13). For example, Cas13a of the present disclosure can be activated by a target RNA to initiate trans cleavage activity of the Cas13a for the cleavage of an RNA reporter and can be activated by a target DNA to initiate trans cleavage activity of the Cas13a for trans cleavage of an RNA reporter. An RNA reporter can be an RNA-based reporter molecule. In some embodiments, the Cas13a recognizes and detects ssDNA to initiate transcleavage of RNA reporters. Multiple Cas13a isolates can recognize, be activated by, and detect target DNA, including ssDNA, upon hybridization of a guide nucleic acid with the target DNA. For example, LbuCas13a and LwaCas13a can both be activated to transcollaterally cleave RNA reporters by target DNA. Thus, Type VI CRISPR/Cas enzyme (e.g., Cas13, such as Cas13a) can be DNA-activated programmable RNA nucleases, and therefore, can be used to detect a target DNA using the methods as described herein. DNA-activated programmable RNA nuclease detection of ssDNA can be robust at multiple pH values. For example, target ssDNA detection by Cas13 can exhibit consistent cleavage across a wide range of pH conditions, such as from a pH of 6.8 to a pH of 8.2. In contrast, target RNA detection by Cas13 may exhibit high cleavage activity of pH values from 7.9 to 8.2. In some embodiments, a DNA-activated programmable RNA nuclease that also is capable of being an RNA-activated programmable RNA nuclease, can have DNA targeting preferences that are distinct from its RNA targeting preferences. For example, the optimal ssDNA targets for Cas13a have different properties than optimal RNA targets for Cas13a. As one example, gRNA performance on ssDNA may not necessarily correlate with the performance of the same gRNAs on RNA. As another example, gRNAs can perform at a high level regardless of target nucleotide identity at a 3′ position on a target RNA sequence. In some embodiments, gRNAs can perform at a high level in the absence of a G at a 3′ position on a target ssDNA sequence. Furthermore, target DNA detected by Cas13 disclosed herein can be directly from organisms, or can be indirectly generated by nucleic acid amplification methods, such as PCR and LAMP or any amplification method described herein. Key steps for the sensitive detection of a target DNA, such as a target ssDNA, by a DNA-activated programmable RNA nuclease, such as Cas13a, can include: (1) production or isolation of DNA to concentrations above about 0.1 nM per reaction for in vitro diagnostics, (2) selection of a target sequence with the appropriate sequence features to enable DNA detection as these features are distinct from those required for RNA detection, and (3) buffer composition that enhances DNA detection. The detection of a target DNA by a DNA-activated programmable RNA nuclease can be connected to a variety of readouts including fluorescence, lateral flow, electrochemistry, or any other readouts described herein. Multiplexing of programmable DNA nuclease, such as a Type V CRISPR-Cas protein, with a DNA-activated programmable RNA nuclease, such as a Type VI protein, with a DNA reporter and an RNA reporter, can enable multiplexed detection of target ssDNAs or a combination of a target dsDNA and a target ssDNA, respectively. Multiplexing of different RNA-activated programmable RNA nucleases that have distinct RNA reporter cleavage preferences can enable additional multiplexing. Methods for the generation of ssDNA for DNA-activated programmable RNA nuclease-based diagnostics can include (1) asymmetric PCR, (2) asymmetric isothermal amplification, such as RPA, LAMP, SDA, etc. (3) NEAR for the production of short ssDNA molecules, and (4) conversion of RNA targets into ssDNA by a reverse transcriptase followed by RNase H digestion. Thus, DNA-activated programmable RNA nuclease detection of target DNA is compatible with the various systems, kits, compositions, reagents, and methods disclosed herein. For example target ssDNA detection by Cas13a can be employed in a DETECTR assay disclosed herein.
Described herein are reagents comprising a single stranded detector nucleic acid comprising a detection moiety, wherein the detector nucleic acid is capable of being cleaved by the activated nuclease, thereby generating a first detectable signal. As used herein, a detector nucleic acid is used interchangeably with reporter or reporter molecule. In some cases, the detector nucleic acid is a single-stranded nucleic acid comprising deoxyribonucleotides. In other cases, the detector nucleic acid is a single-stranded nucleic acid comprising ribonucleotides. The detector nucleic acid can be a single-stranded nucleic acid comprising at least one deoxyribonucleotide and at least one ribonucleotide. In some cases, the detector nucleic acid is a single-stranded nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site. In some cases, the detector nucleic acid comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ribonucleotide residues at an internal position. Sometimes the ribonucleotide residues are continuous. Alternatively, the ribonucleotide residues are interspersed in between non-ribonucleotide residues. In some cases, the detector nucleic acid has only ribonucleotide residues. In some cases, the detector nucleic acid has only deoxyribonucleotide residues. In some cases, the detector nucleic acid comprises nucleotides resistant to cleavage by the programmable nuclease described herein. In some cases, the detector nucleic acid comprises synthetic nucleotides. In some cases, the detector nucleic acid comprises at least one ribonucleotide residue and at least one non-ribonucleotide residue. In some cases, detector nucleic acid is 5-20, 5-15, 5-10, 7-20, 7-15, or 7-10 nucleotides in length. In some cases, the detector nucleic acid comprises at least one uracil ribonucleotide. In some cases, the detector nucleic acid comprises at least two uracil ribonucleotides. Sometimes the detector nucleic acid has only uracil ribonucleotides. In some cases, the detector nucleic acid comprises at least one adenine ribonucleotide. In some cases, the detector nucleic acid comprises at least two adenine ribonucleotide. In some cases, the detector nucleic acid has only adenine ribonucleotides. In some cases, the detector nucleic acid comprises at least one cytosine ribonucleotide. In some cases, the detector nucleic acid comprises at least two cytosine ribonucleotide. In some cases, the detector nucleic acid comprises at least one guanine ribonucleotide. In some cases, the detector nucleic acid comprises at least two guanine ribonucleotide. A detector nucleic acid can comprise only unmodified ribonucleotides, only unmodified deoxyribonucleotides, or a combination thereof. In some cases, the detector nucleic acid is from 5 to 12 nucleotides in length. In some cases, the detector nucleic acid is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, the detector nucleic acid is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. For cleavage by a programmable nuclease comprising Cas13, a detector nucleic acid can be 5, 8, or 10 nucleotides in length. For cleavage by a programmable nuclease comprising Cas12, a detector nucleic acid can be 10 nucleotides in length.
The single stranded detector nucleic acid comprises a detection moiety capable of generating a first detectable signal. Sometimes the detector nucleic acid comprises a protein capable of generating a signal. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorometric, etc.), or piezo-electric signal. In some cases, a detection moiety is on one side of the cleavage site. Optionally, a quenching moiety is on the other side of the cleavage site. Sometimes the quenching moiety is a fluorescence quenching moiety. In some cases, the quenching moiety is 5′ to the cleavage site and the detection moiety is 3′ to the cleavage site. In some cases, the detection moiety is 5′ to the cleavage site and the quenching moiety is 3′ to the cleavage site. Sometimes the quenching moiety is at the 5′ terminus of the detector nucleic acid. Sometimes the detection moiety is at the 3′ terminus of the detector nucleic acid. In some cases, the detection moiety is at the 5′ terminus of the detector nucleic acid. In some cases, the quenching moiety is at the 3′ terminus of the detector nucleic acid. In some cases, the single-stranded detector nucleic acid is at least one population of the single-stranded nucleic acid capable of generating a first detectable signal. In some cases, the single-stranded detector nucleic acid is a population of the single stranded nucleic acid capable of generating a first detectable signal. Optionally, there is more than one population of single-stranded detector nucleic acid. In some cases, there are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, or greater than 50, or any number spanned by the range of this list of different populations of single-stranded detector nucleic acids capable of generating a detectable signal.
A detection moiety can be an infrared fluorophore. A detection moiety can be a fluorophore that emits fluorescence in the range of from 500 nm and 720 nm. A detection moiety can be a fluorophore that emits fluorescence in the range of from 500 nm and 720 nm. In some cases, the detection moiety emits fluorescence at a wavelength of 700 nm or higher. In other cases, the detection moiety emits fluorescence at about 660 nm or about 670 nm. In some cases, the detection moiety emits fluorescence at in the range of from 500 to 520, 500 to 540, 500 to 590, 590 to 600, 600 to 610, 610 to 620, 620 to 630, 630 to 640, 640 to 650, 650 to 660, 660 to 670, 670 to 680, 6890 to 690, 690 to 700, 700 to 710, 710 to 720, or 720 to 730 nm. A detection moiety can be a fluorophore that emits a fluorescence in the same range as 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor, or ATTO TM 633 (NHS Ester). A detection moiety can be fluorescein amidite, 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHS Ester). A detection moiety can be a fluorophore that emits a fluorescence in the same range as 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies). A detection moiety can be fluorescein amidite, 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies). Any of the detection moieties described herein can be from any commercially available source, can be an alternative with a similar function, a generic, or a non-tradename of the detection moieties listed.
A detection moiety can be chosen for use based on the type of sample to be tested. For example, a detection moiety that is an infrared fluorophore is used with a urine sample. As another example, SEQ ID NO: 1 with a fluorophore that emits around 520 nm is used for testing in non-urine samples, and SEQ ID NO: 8 with a fluorophore that emits a fluorescence around 700 nm is used for testing in urine samples.
A quenching moiety can be chosen based on its ability to quench the detection moiety. A quenching moiety can be a non-fluorescent fluorescence quencher. A quenching moiety can quench a detection moiety that emits fluorescence in the range of from 500 nm and 720 nm. A quenching moiety can quench a detection moiety that emits fluorescence in the range of from 500 nm and 720 nm. In some cases, the quenching moiety quenches a detection moiety that emits fluorescence at a wavelength of 700 nm or higher. In other cases, the quenching moiety quenches a detection moiety that emits fluorescence at about 660 nm or about 670 nm. In some cases, the quenching moiety quenches a detection moiety emits fluorescence at in the range of from 500 to 520, 500 to 540, 500 to 590, 590 to 600, 600 to 610, 610 to 620, 620 to 630, 630 to 640, 640 to 650, 650 to 660, 660 to 670, 670 to 680, 6890 to 690, 690 to 700, 700 to 710, 710 to 720, or 720 to 730 nm. A quenching moiety can quench fluorescein amidite, 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHS Ester). A quenching moiety can be Iowa Black RQ, Iowa Black FQ or IRDye QC-1 Quencher. A quenching moiety can quench fluorescein amidite, 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies). A quenching moiety can be Iowa Black RQ (Integrated DNA Technologies), Iowa Black FQ (Integrated DNA Technologies) or IRDye QC-1 Quencher (LiCor). Any of the quenching moieties described herein can be from any commercially available source, can be an alternative with a similar function, a generic, or a non-tradename of the quenching moieties listed.
The generation of the detectable signal from the release of the detection moiety indicates that cleavage by the programmable nuclease has occurred and that the sample contains the target nucleic acid. In some cases, the detection moiety comprises a fluorescent dye. Sometimes the detection moiety comprises a fluorescence resonance energy transfer (FRET) pair. In some cases, the detection moiety comprises an infrared (IR) dye. In some cases, the detection moiety comprises an ultraviolet (UV) dye. Alternatively or in combination, the detection moiety comprises a polypeptide. Sometimes the detection moiety comprises a biotin. Sometimes the detection moiety comprises at least one of avidin or streptavidin. In some instances, the detection moiety comprises a polysaccharide, a polymer, or a nanoparticle. In some instances, the detection moiety comprises a gold nanoparticle or a latex nanoparticle.
A detection moiety can be any moiety capable of generating a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. A detector nucleic acid, sometimes, is protein-nucleic acid that is capable of generating a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal upon cleavage of the nucleic acid. A protein-nucleic acid may comprise a nucleic acid component and a protein or peptide component. In some embodiments, a protein-nucleic acid may comprise a nucleic acid fused to a protein or peptide. Often a calorimetric signal is heat produced after cleavage of the detector nucleic acids. Sometimes, a calorimetric signal is heat absorbed after cleavage of the detector nucleic acids. A potentiometric signal, for example, is electrical potential produced after cleavage of the detector nucleic acids. An amperometric signal can be movement of electrons produced after the cleavage of detector nucleic acid. Often, the signal is an optical signal, such as a colorimetric signal or a fluorescence signal. An optical signal is, for example, a light output produced after the cleavage of the detector nucleic acids. Sometimes, an optical signal is a change in light absorbance between before and after the cleavage of detector nucleic acids. Often, a piezo-electric signal is a change in mass between before and after the cleavage of the detector nucleic acid.
Often, the protein-nucleic acid is an enzyme-nucleic acid. The enzyme may be sterically hindered when present as in the enzyme-nucleic acid, but then functional upon cleavage from the nucleic acid. Often, the enzyme is an enzyme that produces a reaction with a substrate. An enzyme can be invertase. Often, the substrate of invertase is sucrose and DNS reagent.
Sometimes the protein-nucleic acid is a substrate-nucleic acid. Often the substrate is a substrate that produces a reaction with an enzyme.
A protein-nucleic acid may be attached to a solid support. The solid support, for example, is a surface. A surface can be an electrode. Sometimes the solid support is a bead. Often the bead is a magnetic bead. Upon cleavage, the protein is liberated from the solid and interacts with other mixtures. For example, the protein is an enzyme, and upon cleavage of the nucleic acid of the enzyme-nucleic acid, the enzyme flows through a chamber into a mixture comprising the substrate. When the enzyme meets the enzyme substrate, a reaction occurs, such as a colorimetric reaction, which is then detected. As another example, the protein is an enzyme substrate, and upon cleavage of the nucleic acid of the enzyme substrate-nucleic acid, the enzyme flows through a chamber into a mixture comprising the enzyme. When the enzyme substrate meets the enzyme, a reaction occurs, such as a calorimetric reaction, which is then detected.
In some embodiments, the reporter comprises a nucleic acid conjugated to an affinity molecule and the affinity molecule conjugated to the fluorophore (e.g., nucleic acid—affinity molecule—fluorophore) or the nucleic acid conjugated to the fluorophore and the fluorophore conjugated to the affinity molecule (e.g., nucleic acid—fluorophore—affinity molecule). In some embodiments, a linker conjugates the nucleic acid to the affinity molecule. In some embodiments, a linker conjugates the affinity molecule to the fluorophore. In some embodiments, a linker conjugates the nucleic acid to the fluorophore. A linker can be any suitable linker known in the art. In some embodiments, the nucleic acid of the reporter can be directly conjugated to the affinity molecule and the affinity molecule can be directly conjugated to the fluorophore or the nucleic acid can be directly conjugated to the fluorophore and the fluorophore can be directly conjugated to the affinity molecule. In this context, “directly conjugated” indicated that no intervening molecules, polypeptides, proteins, or other moieties are present between the two moieties directly conjugated to each other. For example, if a reporter comprises a nucleic acid directly conjugated to an affinity molecule and an affinity molecule directly conjugated to a fluorophore—no intervening moiety is present between the nucleic acid and the affinity molecule and no intervening moiety is present between the affinity molecule and the fluorophore. The affinity molecule can be biotin, avidin, streptavidin, or any similar molecule.
In some cases, the reporter comprises a substrate-nucleic acid. The substrate may be sequestered from its cognate enzyme when present as in the substrate-nucleic acid, but then is released from the nucleic acid upon cleavage, wherein the released substrate can contact the cognate enzyme to produce a detectable signal. Often, the substrate is sucrose and the cognate enzyme is invertase, and a DNS reagent can be used to monitor invertase activity.
A major advantage of the devices and methods disclosed herein is the design of excess reporters to total nucleic acids in an unamplified or an amplified sample, not including the nucleic acid of the reporter. Total nucleic acids can include the target nucleic acids and non-target nucleic acids, not including the nucleic acid of the reporter. The non-target nucleic acids can be from the original sample, either lysed or unlysed. The non-target nucleic acids can also be byproducts of amplification. Thus, the non-target nucleic acids can include both non-target nucleic acids from the original sample, lysed or unlysed, and from an amplified sample. The presence of a large amount of non-target nucleic acids, an activated programmable nuclease may be inhibited in its ability to bind and cleave the reporter sequences. This is because the activated programmable nucleases collaterally cleaves any nucleic acids. If total nucleic acids are in present in large amounts, they may outcompete reporters for the programmable nucleases. The devices and methods disclosed herein are designed to have an excess of reporter to total nucleic acids, such that the detectable signals from cleavage reactions (e.g., DETECTR reactions) are particularly superior. In some embodiments, the reporter can be present in at least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, from 1.5 fold to 100 fold, from 2 fold to 10 fold, from 10 fold to 20 fold, from 20 fold to 30 fold, from 30 fold to 40 fold, from 40 fold to 50 fold, from 50 fold to 60 fold, from 60 fold to 70 fold, from 70 fold to 80 fold, from 80 fold to 90 fold, from 90 fold to 100 fold, from 1.5 fold to 10 fold, from 1.5 fold to 20 fold, from 10 fold to 40 fold, from 20 fold to 60 fold, or from 10 fold to 80 fold excess of total nucleic acids.
A second significant advantage of the devices and methods disclosed herein is the design of an excess volume comprising the guide nucleic acid, the programmable nuclease, and the reporter, which contacts a smaller volume comprising the sample with the target nucleic acid of interest. The smaller volume comprising the sample can be unlysed sample, lysed sample, or lysed sample which has undergone any combination of reverse transcription, amplification, and in vitro transcription. The presence of various reagents in a crude, non-lysed sample, a lysed sample, or a lysed and amplified sample, such as buffer, magnesium sulfate, salts, the pH, a reducing agent, primers, dNTPs, NTPs, cellular lysates, non-target nucleic acids, primers, or other components, can inhibit the ability of the programmable nuclease to find and cleave the nucleic acid of the reporter. This may be due to nucleic acids that are not the reporter, which outcompete the nucleic acid of the reporter, for the programmable nuclease. Alternatively, various reagents in the sample may simply inhibit the activity of the programmable nuclease. Thus, the devices and methods provided herein for contacting an excess volume comprising the guide nucleic acid, the programmable nuclease, and the reporter to a smaller volume comprising the sample with the target nucleic acid of interest provides for superior detection of the target nucleic acid by ensuring that the programmable nuclease is able to find and cleaves the nucleic acid of the reporter. In some embodiments, the volume comprising the guide nucleic acid, the programmable nuclease, and the reporter (can be referred to as “a second volume”) is 4-fold greater than a volume comprising the sample (can be referred to as “a first volume”). In some embodiments, the volume comprising the guide nucleic acid, the programmable nuclease, and the reporter (can be referred to as “a second volume”) is at least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, from 1.5 fold to 100 fold, from 2 fold to 10 fold, from 10 fold to 20 fold, from 20 fold to 30 fold, from 30 fold to 40 fold, from 40 fold to 50 fold, from 50 fold to 60 fold, from 60 fold to 70 fold, from 70 fold to 80 fold, from 80 fold to 90 fold, from 90 fold to 100 fold, from 1.5 fold to 10 fold, from 1.5 fold to 20 fold, from 10 fold to 40 fold, from 20 fold to 60 fold, or from 10 fold to 80 fold greater than a volume comprising the sample (can be referred to as “a first volume”). In some embodiments, the volume comprising the sample is at least 0.5 ul, at least 1 ul, at least at least 1 uL, at least 2 uL, at least 3 uL, at least 4 uL, at least 5 uL, at least 6 uL, at least 7 uL, at least 8 uL, at least 9 uL, at least 10 uL, at least 11 uL, at least 12 uL, at least 13 uL, at least 14 uL, at least 15 uL, at least 16 uL, at least 17 uL, at least 18 uL, at least 19 uL, at least 20 uL, at least 25 uL, at least 30 uL, at least 35 uL, at least 40 uL, at least 45 uL, at least 50 uL, at least 55 uL, at least 60 uL, at least 65 uL, at least 70 uL, at least 75 uL, at least 80 uL, at least 85 uL, at least 90 uL, at least 95 uL, at least 100 uL, from 0.5 uL to 5 ul uL, from 5 uL to 10 uL, from 10 uL to 15 uL, from 15 uL to 20 uL, from 20 uL to 25 uL, from 25 uL to 30 uL, from 30 uL to 35 uL, from 35 uL to 40 uL, from 40 uL to 45 uL, from 45 uL to 50 uL, from 10 uL to 20 uL, from 5 uL to 20 uL, from 1 uL to 40 uL, from 2 uL to 10 uL, or from 1 uL to 10 uL. In some embodiments, the volume comprising the programmable nuclease, the guide nucleic acid, and the reporter is at least 10 uL, at least 11 uL, at least 12 uL, at least 13 uL, at least 14 uL, at least 15 uL, at least 16 uL, at least 17 uL, at least 18 uL, at least 19 uL, at least 20 uL, at least 21 uL, at least 22 uL, at least 23 uL, at least 24 uL, at least 25 uL, at least 26 uL, at least 27 uL, at least 28 uL, at least 29 uL, at least 30 uL, at least 40 uL, at least 50 uL, at least 60 uL, at least 70 uL, at least 80 uL, at least 90 uL, at least 100 uL, at least 150 uL, at least 200 uL, at least 250 uL, at least 300 uL, at least 350 uL, at least 400 uL, at least 450 uL, at least 500 uL, from 10 uL to 15 ul uL, from 15 uL to 20 uL, from 20 uL to 25 uL, from 25 uL to 30 uL, from 30 uL to 35 uL, from 35 uL to 40 uL, from 40 uL to 45 uL, from 45 uL to 50 uL, from 50 uL to 55 uL, from 55 uL to 60 uL, from 60 uL to 65 uL, from 65 uL to 70 uL, from 70 uL to 75 uL, from 75 uL to 80 uL, from 80 uL to 85 uL, from 85 uL to 90 uL, from 90 uL to 95 uL, from 95 uL to 100 uL, from 100 uL to 150 uL, from 150 uL to 200 uL, from 200 uL to 250 uL, from 250 uL to 300 uL, from 300 uL to 350 uL, from 350 uL to 400 uL, from 400 uL to 450 uL, from 450 uL to 500 uL, from 10 uL to 20 uL, from 10 uL to 30 uL, from 25 uL to 35 uL, from 10 uL to 40 uL, from 20 uL to 50 uL, from 18 uL to 28 uL, or from 17 uL to 22 uL.
A reporter may be a hybrid nucleic acid reporter. A hybrid nucleic acid reporter comprises a nucleic acid with at least one deoxyribonucleotide and at least one ribonucleotide. In some embodiments, the nucleic acid of the hybrid nucleic acid reporter can be of any length and can have any mixture of DNAs and RNAs. For example, in some cases, longer stretches of DNA can be interrupted by a few ribonucleotides. Alternatively, longer stretches of RNA can be interrupted by a few deoxyribonucleotides. Alternatively, every other base in the nucleic acid may alternate between ribonucleotides and deoxyribonucleotides. A major advantage of the hybrid nucleic acid reporter is increased stability as compared to a pure RNA nucleic acid reporter. For example, a hybrid nucleic acid reporter can be more stable in solution, lyophilized, or vitrified as compared to a pure DNA or pure RNA reporter.
The reporter can be lyophilized or vitrified. The reporter can be suspended in solution or immobilized on a surface. For example, the reporter can be immobilized on the surface of a chamber in a device as disclosed herein. In some cases, the reporter is immobilized on beads, such as magnetic beads, in a chamber of a device as disclosed herein where they are held in position by a magnet placed below the chamber.
Additionally, target nucleic acid can be amplified before binding to the crRNA of the CRISPR enzyme. This amplification can be PCR amplification or isothermal amplification. This nucleic acid amplification of the sample can improve at least one of sensitivity, specificity, or accuracy of the detection the target RNA. The reagents for nucleic acid amplification can comprise a recombinase, an oligonucleotide primer, a single-stranded DNA binding (SSB) protein, and a polymerase. The nucleic acid amplification can be transcription mediated amplification (TMA). Nucleic acid amplification can be helicase dependent amplification (HDA) or circular helicase dependent amplification (cHDA). In additional cases, nucleic acid amplification is strand displacement amplification (SDA). The nucleic acid amplification can be recombinase polymerase amplification (RPA). The nucleic acid amplification can be at least one of loop mediated amplification (LAMP) or the exponential amplification reaction (EXPAR). Nucleic acid amplification is, in some cases, by rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence based amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), or improved multiple displacement amplification (IMDA). The nucleic acid amplification can be performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes. Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 20-45° C. The nucleic acid amplification reaction can be performed at a temperature no greater than 20° C., 25° C., 30° C., 35° C., 37° C., 40° C., 45° C. The nucleic acid amplification reaction can be performed at a temperature of at least 20° C., 25° C., 30° C., 35° C., 37° C., 40° C., or 45° C.
Disclosed herein are methods of assaying for a target nucleic acid as described herein wherein a signal is detected. For example, a method of assaying for a target nucleic acid in a sample comprises contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; and assaying for a signal indicating cleavage of at least some protein-nucleic acids of a population of protein-nucleic acids, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. As another example, a method of assaying for a target nucleic acid in a sample, for example, comprises: a) contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; b) contacting the complex to a substrate; c) contacting the substrate to a reagent that differentially reacts with a cleaved substrate; and d) assaying for a signal indicating cleavage of the substrate, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. Often, the substrate is an enzyme-nucleic acid. Sometimes, the substrate is an enzyme substrate-nucleic acid.
A programmable nuclease can comprise a programmable nuclease capable of being activated when complexed with a guide nucleic acid and target nucleic acid. The programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic acid, in which the activated programmable nuclease can cleave the target nucleic acid and can have trans cleavage activity. Trans cleavage activity can be non-specific cleavage of nearby nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety. Once the detector nucleic acid is cleaved by the activated programmable nuclease, the detection moiety can be released from the detector nucleic acid and can generate a signal. The signal can be immobilized on a support medium for detection. The signal can be visualized to assess whether a target nucleic acid comprises a modification.
Often, the signal is a colorimetric signal or a signal visible by eye. In some instances, the signal is fluorescent, electrical, chemical, electrochemical, or magnetic. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorometric, etc.), or piezo-electric signal. In some cases, the detectable signal is a colorimetric signal or a signal visible by eye. In some instances, the detectable signal is fluorescent, electrical, chemical, electrochemical, or magnetic. In some cases, the first detection signal is generated by binding of the detection moiety to the capture molecule in the detection region, where the first detection signal indicates that the sample contained the target nucleic acid. Sometimes the system is capable of detecting more than one type of target nucleic acid, wherein the system comprises more than one type of guide nucleic acid and more than one type of detector nucleic acid. In some cases, the detectable signal is generated directly by the cleavage event. Alternatively or in combination, the detectable signal is generated indirectly by the signal event. Sometimes the detectable signal is not a fluorescent signal. In some instances, the detectable signal is a colorimetric or color-based signal. In some cases, the detected target nucleic acid is identified based on its spatial location on the detection region of the support medium. In some cases, the second detectable signal is generated in a spatially distinct location than the first generated signal.
In some cases, the threshold of detection, for a subject method of detecting a single stranded target nucleic acid in a sample, is less than or equal to 10 nM. The term “threshold of detection” is used herein to describe the minimal amount of target nucleic acid that must be present in a sample in order for detection to occur. For example, when a threshold of detection is 10 nM, then a signal can be detected when a target nucleic acid is present in the sample at a concentration of 10 nM or more. In some cases, the threshold of detection is less than or equal to 5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, 0.005 nM, 0.001 nM, 0.0005 nM, 0.0001 nM, 0.00005 nM, 0.00001 nM, 10 pM, 1 pM, 500 fM, 250 fM, 100 fM, 50 fM, 10 fM, 5 fM, 1 fM, 500 attomole (aM), 100 aM, 50 aM, 10 aM, or 1 aM. In some cases, the threshold of detection is in a range of from 1 aM to 1 nM, 1 aM to 500 pM, 1 aM to 200 pM, 1 aM to 100 pM, 1 aM to 10 pM, 1 aM to 1 pM, 1 aM to 500 fM, 1 aM to 100 fM, 1 aM to 1 fM, 1 aM to 500 aM, 1 aM to 100 aM, 1 aM to 50 aM, 1 aM to 10 aM, 10 aM to 1 nM, 10 aM to 500 pM, 10 aM to 200 pM, 10 aM to 100 pM, 10 aM to 10 pM, 10 aM to 1 pM, 10 aM to 500 fM, 10 aM to 100 fM, 10 aM to 1 fM, 10 aM to 500 aM, 10 aM to 100 aM, 10 aM to 50 aM, 100 aM to 1 nM, 100 aM to 500 pM, 100 aM to 200 pM, 100 aM to 100 pM, 100 aM to 10 pM, 100 aM to 1 pM, 100 aM to 500 fM, 100 aM to 100 fM, 100 aM to 1 fM, 100 aM to 500 aM, 500 aM to 1 nM, 500 aM to 500 pM, 500 aM to 200 pM, 500 aM to 100 pM, 500 aM to 10 pM, 500 aM to 1 pM, 500 aM to 500 fM, 500 aM to 100 fM, 500 aM to 1 fM, 1 fM to 1 nM, 1 fM to 500 pM, 1 fM to 200 pM, 1 fM to 100 pM, 1 fM to 10 pM, 1 fM to 1 pM, 10 fM to 1 nM, 10 fM to 500 pM, 10 fM to 200 pM, 10 fM to 100 pM, 10 fM to 10 pM, 10 fM to 1 pM, 500 fM to 1 nM, 500 fM to 500 pM, 500 fM to 200 pM, 500 fM to 100 pM, 500 fM to 10 pM, 500 fM to 1 pM, 800 fM to 1 nM, 800 fM to 500 pM, 800 fM to 200 pM, 800 fM to 100 pM, 800 fM to 10 pM, 800 fM to 1 pM, fom 1 pM to 1 nM, 1 pM to 500 pM, 1 pM to 200 pM, 1 pM to 100 pM, or 1 pM to 10 pM. In some cases, the threshold of detection in a range of from 800 fM to 100 pM, 1 pM to 10 pM, 10 fM to 500 fM, 10 fM to 50 fM, 50 fM to 100 fM, 100 fM to 250 fM, or 250 fM to 500 fM. In some cases, the minimum concentration at which a single stranded target nucleic acid is detected in a sample is in a range of from 1 aM to 1 nM, 10 aM to 1 nM, 100 aM to 1 nM, 500 aM to 1 nM, 1 fM to 1 nM, 1 fM to 500 pM, 1 fM to 200 pM, 1 fM to 100 pM, 1 fM to 10 pM, 1 fM to 1 pM, 10 fM to 1 nM, 10 fM to 500 pM, 10 fM to 200 pM, 10 fM to 100 pM, 10 fM to 10 pM, 10 fM to 1 pM, 500 fM to 1 nM, 500 fM to 500 pM, 500 fM to 200 pM, 500 fM to 100 pM, 500 fM to 10 pM, 500 fM to 1 pM, 800 fM to 1 nM, 800 fM to 500 pM, 800 fM to 200 pM, 800 fM to 100 pM, 800 fM to 10 pM, 800 fM to 1 pM, 1 pM to 1 nM, 1 pM to 500 pM, from 1 pM to 200 pM, 1 pM to 100 pM, or 1 pM to 10 pM. In some cases, the minimum concentration at which a single stranded target nucleic acid can be detected in a sample is in a range of from 1 aM to 100 pM. In some cases, the minimum concentration at which a single stranded target nucleic acid can be detected in a sample is in a range of from 1 fM to 100 pM. In some cases, the minimum concentration at which a single stranded target nucleic acid can be detected in a sample is in a range of from 10 fM to 100 pM. In some cases, the minimum concentration at which a single stranded target nucleic acid can be detected in a sample is in a range of from 800 fM to 100 pM. In some cases, the minimum concentration at which a single stranded target nucleic acid can be detected in a sample is in a range of from 1 pM to 10 pM. In some cases, the devices, systems, fluidic devices, kits, and methods described herein detect a target single-stranded nucleic acid in a sample comprising a plurality of nucleic acids such as a plurality of non-target nucleic acids, where the target single-stranded nucleic acid is present at a concentration as low as 1 aM, 10 aM, 100 aM, 500 aM, 1 fM, 10 fM, 500 fM, 800 fM, 1 pM, 10 pM, 100 pM, or 1 pM.
In some cases, the devices, systems, fluidic devices, kits, and methods described herein detect a target single-stranded nucleic acid in a sample where the sample is contacted with the reagents for a predetermined length of time sufficient for the trans cleavage to occur or cleavage reaction to reach completion. In some cases, the devices, systems, fluidic devices, kits, and methods described herein detect a target single-stranded nucleic acid in a sample where the sample is contacted with the reagents for no greater than 60 minutes. Sometimes the sample is contacted with the reagents for no greater than 120 minutes, 110 minutes, 100 minutes, 90 minutes, 80 minutes, 70 minutes, 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute. Sometimes the sample is contacted with the reagents for at least 120 minutes, 110 minutes, 100 minutes, 90 minutes, 80 minutes, 70 minutes, 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes. In some cases, the devices, systems, fluidic devices, kits, and methods described herein can detect a target nucleic acid in a sample in less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, less than 50 minutes, less than 45 minutes, less than 40 minutes, less than 35 minutes, less than 30 minutes, less than 25 minutes, less than 20 minutes, less than 15 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, or less than 5 minutes.
When a guide nucleic acid binds to a target nucleic acid, the programmable nuclease's trans cleavage activity can be initiated, and detector nucleic acids can be cleaved, resulting in the detection of fluorescence. Some methods as described herein can a method of assaying for a target nucleic acid in a sample comprises contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; and assaying for a signal indicating cleavage of at least some protein-nucleic acids of a population of protein-nucleic acids, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. The cleaving of the detector nucleic acid using the programmable nuclease may cleave with an efficiency of 50% as measured by a change in a signal that is calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric, as non-limiting examples. Some methods as described herein can be a method of detecting a target nucleic acid in a sample comprising contacting the sample comprising the target nucleic acid with a guide nucleic acid targeting a target nucleic acid segment, a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment, a single stranded detector nucleic acid comprising a detection moiety, wherein the detector nucleic acid is capable of being cleaved by the activated programmable nuclease, thereby generating a first detectable signal, cleaving the single stranded detector nucleic acid using the programmable nuclease that cleaves as measured by a change in color, and measuring the first detectable signal on the support medium. The cleaving of the single stranded detector nucleic acid using the programmable nuclease may cleave with an efficiency of 50% as measured by a change in color. In some cases, the cleavage efficiency is at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% as measured by a change in color. The change in color may be a detectable colorimetric signal or a signal visible by eye. The change in color may be measured as a first detectable signal. The first detectable signal can be detectable within 5 minutes of contacting the sample comprising the target nucleic acid with a guide nucleic acid targeting a target nucleic acid segment, a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment, and a single stranded detector nucleic acid comprising a detection moiety, wherein the detector nucleic acid is capable of being cleaved by the activated nuclease. The first detectable signal can be detectable within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, or 120 minutes of contacting the sample.
In some cases, the devices, systems, fluidic devices, kits, and methods described herein detect a target single-stranded nucleic acid with a programmable nuclease and a single-stranded detector nucleic acid in a sample where the sample is contacted with the reagents for a predetermined length of time sufficient for trans cleavage of the single stranded detector nucleic acid. For example, a programmable nuclease is LbuCas13a that detects a target nucleic acid and a single stranded detector nucleic acid comprises two adjacent uracil nucleotides with a green detectable moiety that is detected upon cleavage. As another example, a programmable nuclease is LbaCas13a that detects a target nucleic acid and a single-stranded detector nucleic acid comprises two adjacent adenine nucleotides with a red detectable moiety that is detected upon cleavage.
In some cases, the devices, systems, fluidic devices, kits, and methods described herein detect different two target single-stranded nucleic acids with two different programmable nucleases and two different single-stranded detector nucleic acids in a sample where the sample is contacted with the reagents for a predetermined length of time sufficient for trans cleavage of the at least two single-stranded detector nucleic acids. For example, a first programmable nuclease is LbuCas13a, which is activated by a first single-stranded target nucleic acid and upon activation, cleaves a first single-stranded detector nucleic acid comprising two adjacent uracil nucleotides with a green detectable moiety that is detected upon cleavage, and a second programmable nuclease is LbaCas13a, which is activated by a second single-stranded target nucleic acid and upon activation, cleaves a second single-stranded detector nucleic acid comprising two adjacent adenine nucleotides with a red detectable moiety that is detected upon cleavage. In some cases, the activation of both programmable nucleases to cleave their respective single-stranded nucleic acids, for example LbuCas13a that cleaves a first single-stranded detector nucleic acid comprising two adjacent uracil nucleotides with a green detectable moiety that is detected upon cleavage and LbaCas13a that cleaves a second single-stranded detector nucleic acid comprises two adjacent adenine nucleotides with a red detectable moiety that is detected upon cleavage, the subsequence detection of a yellow signal indicates that the first single-stranded target nucleic acid and the second single-stranded target nucleic are present in the sample.
Alternatively, the devices, systems, fluidic devices, kits, and methods described herein can comprise a first programmable nuclease that detects the presence of a first single-stranded target nucleic acid in a sample and a second programmable nuclease that is used as a control. For example, a first programmable nuclease is Lbu13a, which cleaves a first single-stranded detector nucleic acid comprising two adjacent uracil nucleotides with a green detectable moiety that is detected upon cleavage and which is activated by a first single-stranded target nucleic acid if it is present in the sample, and a second programmable nuclease is Lba13a, which cleaves a second single-stranded detector nucleic acid comprising two adjacent adenine nucleotides with a red detectable moiety that is detected upon cleavage and which is activated by a second single-stranded target nucleic acid that is not found (and would not be expected to ever be found) in the sample and serves as a control. In this case, the detection of a red signal or a yellow signal indicates there is a problem with the test (e.g., the sample contains a high level of other RNAses that are cleaving the single-stranded detector nucleic acids in the absence of activation of the second programmable nuclease), but the detection of a green signal indicates the test is working correctly and the first target single-stranded nucleic acid of the first programmable nuclease is present in the sample.
As additional examples, the devices, systems, fluidic devices, kits, and methods described herein detect different two target single-stranded nucleic acids with two different programmable nucleases and two different single stranded detector nucleic acids in a sample where the sample is contacted with the reagents for a predetermined length of time sufficient for trans cleavage of the at least two single stranded detector nucleic acid. For example, a first programmable nuclease is a Cas13a protein, which cleaves a first single-stranded detector nucleic that is detected upon cleavage and which is activated by a first single-stranded target nucleic acid from a sepsis RNA biomarker if it is present in the sample, and a second programmable nuclease is a Cas14 protein, which cleaves a second single-stranded detector nucleic acid that is detected upon cleavage and which is activated by a second single-stranded target nucleic acid from in influenza virus.
The reagents described herein can also include buffers, which are compatible with the devices, systems, fluidic devices, kits, and methods disclosed herein. These buffers are compatible with the other reagents, samples, and support mediums as described herein for detection of an ailment, such as a disease, including those caused by viruses such as influenza. The methods described herein can also include the use of buffers, which are compatible with the methods disclosed herein. For example, a buffer comprises 20 mM HEPES pH 6.8, 50 mM KCl, 5 mM MgCl2, and 5% glycerol. In some instances the buffer comprises from 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10,5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM HEPES pH 6.8. The buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM KCl. In other instances the buffer comprises 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM MgCl2. The buffer can comprise 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30% glycerol.
As another example, a buffer comprises 100 mM Imidazole pH 7.5; 250 mM KCl, 25 mM MgCl2, 50 ug/mL BSA, 0.05% Igepal Ca-630, and 25% Glycerol. In some instances the buffer comprises 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM Imidazole pH 7.5. The buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM KCl. In other instances the buffer comprises 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM MgCl2. The buffer, in some instances, comprises 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 50, 10 to 75, 10 to 100, 25 to 50, 25 to 75 25 to 100, 50 to 75, or 50 to 100 ug/mL BSA. In some instances, the buffer comprises 0 to 1, 0 to 0.5, 0 to 0.25, 0 to 0.01, 0 to 0.05, 0 to 0.025, 0 to 0.01, 0.01 to 0.025, 0.01 to 0.05, 0.01 to 0.1, 0.01 to 0.25, 0.01, to 0.5, 0.01 to 1, 0.025 to 0.05, 0.025 to 0.1, 0.025, to 0.5, 0.025 to 1, 0.05 to 0.1, 0.05 to 0.25, 0.05 to 0.5, 0.05 to 0.75, 0.05 to 1, 0.1 to 0.25, 0.1 to 0.5, or 0.1 to 1% Igepal Ca-630. The buffer can comprise 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30% glycerol.
A buffer of the present disclosure may comprise a viral lysis buffer. A viral lysis buffer may lyse a coronavirus capsid in a viral sample (e.g., a sample collected from an individual suspected of having a coronavirus infection), releasing a viral genome. The viral lysis buffer may be compatible with amplification (e.g., RT-LAMP amplification) of a target region of the viral genome. The viral lysis buffer may be compatible with detection (e.g., a DETECTR reaction disclosed herein). A viral lysis buffer that is functional to lyse a virus and is compatible with amplification, detection, or both may be a dual lysis buffer. A viral lysis buffer that is functional to lyse a virus and is compatible with amplification may be a dual lysis/amplification buffer. A viral lysis buffer that is functional to lyse a virus and is compatible with detection may be a dual lysis/detection buffer. A sample may be prepared in a one-step sample preparation method comprising suspending the sample in a viral lysis buffer compatible with amplification, detection (e.g., a DETECTR reaction), or both. A viral lysis buffer compatible with amplification (e.g., RT-LAMP amplification), detection (e.g., DETECTR), or both, may comprise a buffer (e.g., Tris-HCl, phosphate, or HEPES), a reducing agent (e.g., N-Acetyl Cysteine (NAC), Dithiothreitol (DTT), β-mercaptoethanol (BME), or tris(2-carboxyethyl)phosphine (TCEP)), a chelating agent (e.g., EDTA or EGTA), a detergent (e.g., deoxycholate, NP-40 (Ipgal), Triton X-100, or Tween 20), a salt (e.g., ammonium acetate, magnesium acetate, manganese acetate, potassium acetate, sodium acetate, ammonium chloride, potassium chloride, magnesium chloride, manganese chloride, sodium chloride, ammonium sulfate, magnesium sulfate, manganese sulfate, potassium sulfate, or sodium sulfate), or a combination thereof. For example, a viral lysis buffer may comprise a buffer and a reducing agent, or a viral lysis buffer may comprise a buffer and a chelating agent. The viral lysis buffer may be formulated at a low pH. For example, the viral lysis buffer may be formulated at a pH of from about pH 4 to about pH 5. In some embodiments, the viral lysis buffer may be formulated at a pH of from about pH 4 to about pH 8.8. In some embodiments, the viral lysis buffer may be formulated at a pH of from about pH 4 to about pH 9. The viral lysis buffer may further comprise a preservative (e.g., ProClin 150). In some embodiments, the viral lysis buffer may comprise an activator of the amplification reaction. For example, the buffer may comprise primers, dNTPs, or magnesium (e.g., MgSO4, MgCl2 or MgOAc), or a combination thereof, to activate the amplification reaction. In some embodiments, an activator (e.g., primers, dNTPs, or magnesium) may be added to the buffer following lysis of the coronavirus to initiate the amplification reaction.
A viral lysis buffer may comprise a pH of about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, or about 9. In some embodiments, a viral lysis buffer may comprise a pH of from 3.5 to 4.5, from 4 to 5, from 4.5 to 5.5, from 3.5 to 4, from 4 to 4.5, from 4.5 to 5, from 5 to 5.5, from 5 to 6, from 6 to 7, from 7 to 8, or from 8 to 9.
A viral lysis buffer may comprise a magnesium concentration of about 0 mM, about 2 mM, about 4 mM, about 5 mM, about 6 mM, about 8 mM, about 10 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, or about 60 mM of magnesium (e.g., MgSO4, MgCl2 or MgOAc). A viral lysis buffer may comprise a magnesium concentration of from 0 mM to 5 mM, from 5 mM to 10 mM, from 10 mM to 15 mM, from 15 mM to 20 mM, from 20 mM to 25 mM, from 25 mM to 30 mM, from 30 mM to 40 mM, from 40 mM to 50 mM, or from 50 mM to 60 mM of magnesium (e.g., MgSO4, MgCl2 or MgOAc). In some embodiments, the magnesium may be added after viral lysis to activate an amplification reaction.
A viral lysis buffer may comprise a reducing agent (e.g., NAC, DTT, BME, or TCEP) at a concentration of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 10 mM, about 12 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 7 mM, about 80 mM, about 90 mM, about 100 mM, or about 120 mM. A viral lysis buffer may comprise a reducing agent (e.g., NAC, DTT, BME, or TCEP) at a concentration of from 1 mM to 5 mM, from 5 mM to 10 mM, from 10 mM to 15 mM, from 15 mM to 20 mM, from 20 mM to 25 mM, or from 25 mM to 30 mM, from 30 mM to 40 mM, from 40 mM to 50 mM, from 50 mM to 60 mM, from 60 mM to 70 mM, from 70 mM to 80 mM, or from 80 mM to 90 mM, from 90 mM to 100 mM, or from 100 mM to 120 mM. A viral lysis buffer may comprise a chelator (e.g., EDTA or EGTA) at a concentration of about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 10 mM, about 12 mM, about 15 mM, about 20 mM, about 25 mM, or about 30 mM. A viral lysis buffer may comprise a chelator (e.g., EDTA or EGTA) at a concentration of from 0.1 mM to 0.5 mM, from 0.25 mM to 0.5 mM, from 0.4 mM to 0.6 mM, from 0.5 mM to 1 mM, from 1 mM to 5 mM, from 5 mM to 10 mM, from 10 mM to 15 mM, from 15 mM to 20 mM, from 20 mM to 25 mM, or from 25 mM to 30 mM.
A viral lysis buffer may comprise a salt (e.g., ammonium acetate ((NH4)2OAc), magnesium acetate (MgOAc), manganese acetate (MnOAc), potassium acetate (K2OAc), sodium acetate (Na2OAc), ammonium chloride (NH4Cl), potassium chloride (KCl), magnesium chloride (MgCl2), manganese chloride (MnCl2), sodium chloride (NaCl), ammonium sulfate ((NH4)2SO4), magnesium sulfate (MgSO4), manganese sulfate (MnSO4), potassium sulfate (K2SO4), or sodium sulfate (Na2SO4)) at a concentration of about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM. A viral lysis buffer may comprise a salt (e.g., (NH4)2OAc, MgOAc, MnOAc, K2OAc, Na2OAc, NH4Cl, KCl, MgCl2, MnCl2, NaCl, (NH4)2SO4, MgSO4, MnSO4, K2SO4, or Na2SO4) at a concentration of from 1 mM to 5 mM, from 1 mM to 10 mM, from 5 mM to 10 mM, from 10 mM to 15 mM, from 15 mM to 20 mM, from 20 mM to 25 mM, from 25 mM to 30 mM, from 30 mM to 35 mM, from 35 mM to 40 mM, from 40 mM to 45 mM, from 45 mM to 50 mM, from 50 mM to 55 mM, from 55 mM to 60 mM, from 60 mM to 70 mM, from 70 mM to 80 mM, from 80 mM to 90 mM, or from 90 mM to 100 mM.
A viral lysis buffer may comprise a detergent (e.g., deoxycholate, NP-40 (Ipgal), Triton X-100, or Tween 20) at a concentration of about 0.01%, about 0.05%, about 0.10%, about 0.15%, about 0.20%, about 0.25%, about 0.30%, about 0.35%, about 0.40%, about 0.45%, about 0.50%, about 0.55%, about 0.60%, about 0.65%, about 0.70%, about 0.75%, about 0.80%, about 0.85%, about 0.90%, about 0.95%, about 1.00%, about 1.10%, about 1.20%, about 1.30%, about 1.40%, about 1.50%, about 2.00%, about 2.50%, about 3.00%, about 3.50%, about 4.00%, about 4.50%, or about 5.00%. A viral lysis buffer may comprise a detergent (e.g., deoxycholate, NP-40 (Ipgal), Triton X-100, or Tween 20) at a concentration of from 0.01% to 0.10%, from 0.05% to 0.15%, from 0.10% to 0.20%, from 0.15% to 0.25%, from 0.20% to 0.30%, from 0.25% to 0.35%, from 0.30% to 0.40%, from 0.35% to 0.45%, from 0.40% to 0.50%, from 0.45% to 0.55%, from 0.50% to 0.60%, from 0.55% to 0.65%, from 0.60% to 0.70%, from 0.65% to 0.75%, from 0.70% to 0.80%, from 0.75% to 0.85%, from 0.80% to 0.90%, from 0.85% to 0.95%, from 0.90% to 1.00%, from 0.95% to 1.10%, from 1.00% to 1.20%, from 1.10% to 1.30%, from 1.20% to 1.40%, from 1.30% to 1.50%, from 1.40% to 1.60%, from 1.50% to 2.00%, from 2.00% to 2.50%, from 2.50% to 3.00%, from 3.00% to 3.50%, from 3.50% to 4.00%, from 4.00% to 4.50%, or from 4.50% to 5.00%.
A lysis reaction may be performed at a range of temperatures. In some embodiments, a lysis reaction may be performed at about room temperature. In some embodiments, a lysis reaction may be performed at about 95° C. In some embodiments, a lysis reaction may be performed at from 1° C. to 10° C., from 4° C. to 8° C., from 10° C. to 20° C., from 15° C. to 25° C., from 15° C. to 20° C., from 18° C. to 25° C., from 18° C. to 95° C., from 20° C. to 37° C., from 25° C. to 40° C., from 35° C. to 45° C., from 40° C. to 60° C., from 50° C. to 70° C., from 60° C. to 80° C., from 70° C. to 90° C., from 80° C. to 95° C., or from 90° C. to 99° C. In some embodiments, a lysis reaction may be performed for about 5 minutes, about 15 minutes, or about 30 minutes. In some embodiments, a lysis reaction may be performed for from 2 minutes to 5 minutes, from 3 minutes to 8 minutes, from 5 minutes to 15 minutes, from 10 minutes to 20 minutes, from 15 minutes to 25 minutes, from 20 minutes to 30 minutes, from 25 minutes to 35 minutes, from 30 minutes to 40 minutes, from 35 minutes to 45 minutes, from 40 minutes to 50 minutes, from 45 minutes to 55 minutes, from 50 minutes to 60 minutes, from 55 minutes to 65 minutes, from 60 minutes to 70 minutes, from 65 minutes to 75 minutes, from 70 minutes to 80 minutes, from 75 minutes to 85 minutes, or from 80 minutes to 90 minutes.
A number of detection devices and methods are consistent with methods disclosed herein. For example, any device that can measure or detect a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorometric, etc.), or piezo-electric signal. Often a calorimetric signal is heat produced after cleavage of the detector nucleic acids. Sometimes, a calorimetric signal is heat absorbed after cleavage of the detector nucleic acids. A potentiometric signal, for example, is electrical potential produced after cleavage of the detector nucleic acids. An amperometric signal can be movement of electrons produced after the cleavage of detector nucleic acid. Often, the signal is an optical signal, such as a colorometric signal or a fluorescence signal. An optical signal is, for example, a light output produced after the cleavage of the detector nucleic acids. Sometimes, an optical signal is a change in light absorbance between before and after the cleavage of detector nucleic acids. Often, a piezo-electric signal is a change in mass between before and after the cleavage of the detector nucleic acid. Sometimes, the detector nucleic acid is protein-nucleic acid. Often, the protein-nucleic acid is an enzyme-nucleic acid.
The results from the detection region from a completed assay can be detected and analyzed in various ways, for example, by a glucometer. In some cases, the positive control spot and the detection spot in the detection region is visible by eye, and the results can be read by the user. In some cases, the positive control spot and the detection spot in the detection region is visualized by an imaging device or other device depending on the type of signal. Often, the imaging device is a digital camera, such a digital camera on a mobile device. The mobile device may have a software program or a mobile application that can capture an image of the support medium, identify the assay being performed, detect the detection region and the detection spot, provide image properties of the detection spot, analyze the image properties of the detection spot, and provide a result. Alternatively or in combination, the imaging device can capture fluorescence, ultraviolet (UV), infrared (IR), or visible wavelength signals. The imaging device may have an excitation source to provide the excitation energy and captures the emitted signals. In some cases, the excitation source can be a camera flash and optionally a filter. In some cases, the imaging device is used together with an imaging box that is placed over the support medium to create a dark room to improve imaging. The imaging box can be a cardboard box that the imaging device can fit into before imaging. In some instances, the imaging box has optical lenses, mirrors, filters, or other optical elements to aid in generating a more focused excitation signal or to capture a more focused emission signal. Often, the imaging box and the imaging device are small, handheld, and portable to facilitate the transport and use of the assay in remote or low resource settings.
The assay described herein can be visualized and analyzed by a mobile application (app) or a software program. Using the graphic user interface (GUI) of the app or program, an individual can take an image of the support medium, including the detection region, barcode, reference color scale, and fiduciary markers on the housing, using a camera on a mobile device. The program or app reads the barcode or identifiable label for the test type, locate the fiduciary marker to orient the sample, and read the detectable signals, compare against the reference color grid, and determine the presence or absence of the target nucleic acid, which indicates the presence of the gene, virus, or the agent responsible for the disease. The mobile application can present the results of the test to the individual. The mobile application can store the test results in the mobile application. The mobile application can communicate with a remote device and transfer the data of the test results. The test results can be viewable remotely from the remote device by another individual, including a healthcare professional. A remote user can access the results and use the information to recommend action for treatment, intervention, clean up of an environment.
Disclosed herein are methods of assaying for a target nucleic acid as described herein that can be used for detection of a mutation in a target nucleic acid. For example, a method of assaying for a target nucleic acid in a sample comprises contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; and assaying for a signal indicating cleavage of at least some protein-nucleic acids of a population of protein-nucleic acids, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. The detection of the signal can indicate the presence of the target nucleic acid. Sometimes, the target nucleic acid comprises a mutation. Often, the mutation is a single nucleotide mutation. As another example, a method of assaying for a target nucleic acid in a sample, for example, comprises: a) contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; b) contacting the complex to a substrate; c) contacting the substrate to a reagent that differentially reacts with a cleaved substrate; and d) assaying for a signal indicating cleavage of the substrate, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. Often, the substrate is an enzyme-nucleic acid. Sometimes, the substrate is an enzyme substrate-nucleic acid.
Methods described herein can be used to identify a mutation in a target nucleic acid. The methods can be used to identify a single nucleotide mutation of a target nucleic acid that affects the expression of a gene. A mutation that affects the expression of gene can be a single nucleotide mutation of a target nucleic acid within the gene, a single nucleotide mutation of a target nucleic acid comprising RNA associated with the expression of a gene, or a target nucleic acid comprising a single nucleotide mutation of a nucleic acid associated with regulation of expression of a gene, such as an RNA or a promoter, enhancer, or repressor of the gene. Often, a status of a mutation is used to diagnose or identify diseases associated with the mutation of target nucleic acid. Detection of target nucleic acids having a mutation are applicable to a number of fields, such as clinically, as a diagnostic, in laboratories as a research tool, and in agricultural applications. Often, the mutation is a single nucleotide mutation. The mutation may result in a mutated strain of a virus, such as an influenza A or influenza B virus.
Disclosed herein are methods of assaying for a target nucleic acid as described herein that can be used for disease detection. For example, a method of assaying for a target nucleic acid (e.g., from an influenza virus) in a sample comprises contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; and assaying for a signal indicating cleavage of at least some protein-nucleic acids of a population of protein-nucleic acids, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. The detection of the signal can indicate the presence of the target nucleic acid. Sometimes, the target nucleic acid comprises a mutation. Often, the mutation is a single nucleotide mutation. As another example, a method of assaying for a target nucleic acid in a sample, for example, comprises: a) contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; b) contacting the complex to a substrate; c) contacting the substrate to a reagent that differentially reacts with a cleaved substrate; and d) assaying for a signal indicating cleavage of the substrate, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. Often, the substrate is an enzyme-nucleic acid. Sometimes, the substrate is an enzyme substrate-nucleic acid.
Methods described herein can be used to identify a mutation in a target nucleic acid from a bacteria, virus, or microbe. The methods can be used to identify a mutation of a target nucleic acid that affects the expression of a gene. A mutation that affects the expression of gene can be a mutation of a target nucleic acid within the gene, a mutation of a target nucleic acid comprising RNA associated with the expression of a gene, or a target nucleic acid comprising a mutation of a nucleic acid associated with regulation of expression of a gene, such as an RNA or a promoter, enhancer, or repressor of the gene. Sometimes, a status of a target nucleic acid mutation is used to determine a pathogenicity of a bacteria, virus, or microbe or treatment resistance, such as resistance to antibiotic treatment. Often, a status of a mutation is used to diagnose or identify diseases associated with the mutation of target nucleic acids in the bacteria, virus, or microbe. Often, the mutation is a single nucleotide mutation.
Disclosed herein are methods of assaying for a target nucleic acid (e.g., from an influenza virus) as described herein that can be used as a research tool, and can be provided as reagent kits. For example, a method of assaying for a target nucleic acid in a sample comprises contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; and assaying for a signal indicating cleavage of at least some protein-nucleic acids of a population of protein-nucleic acids, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. The detection of the signal can indicate the presence of the target nucleic acid. Sometimes, the target nucleic acid comprises a mutation. Often, the mutation is a single nucleotide mutation. As another example, a method of assaying for a target nucleic acid in a sample, for example, comprises: a) contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; b) contacting the complex to a substrate; c) contacting the substrate to a reagent that differentially reacts with a cleaved substrate; and d) assaying for a signal indicating cleavage of the substrate, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. Often, the substrate is an enzyme-nucleic acid. Sometimes, the substrate is an enzyme substrate-nucleic acid.
The methods as described herein can be used to identify a single nucleotide mutation in a target nucleic acid. The methods can be used to identify mutation of a target nucleic acid that affects the expression of a gene. A mutation that affects the expression of gene can be a single nucleotide mutation of a target nucleic acid within the gene, a mutation of a target nucleic acid comprising RNA associated with the expression of a gene, or a target nucleic acid comprising a mutation of a nucleic acid associated with regulation of expression of a gene, such as an RNA or a promoter, enhancer, or repressor of the gene. Often, the mutation is a single nucleotide mutation.
The reagent kits or research tools can be used to detect any number of target nucleic acids, mutations, or other indications disclosed herein in a laboratory setting. Reagent kits can be provided as reagent packs for open box instrumentation.
In other embodiments, any of the systems, assay formats, Cas reporters, programmable nucleases, or other reagents can be used in a point-of-care (POC) test, which can be carried out at a decentralized location such as a hospital, POL, or clinic. These point-of-care tests can be used to diagnose any of the indications disclosed herein, such as influenza or streptococcal infections, or can be used to measure the presence or absence of a particular mutation in a target nucleic acid (e.g., EGFR). POC tests can be provided as small instruments with a consumable test card, wherein the test card is any of the assay formats (e.g., a lateral flow assay) disclosed herein.
In still other embodiments, any of the systems, assay formats, Cas reporters, programmable nucleases, or other reagents can be used in an over-the-counter (OTC), readerless format, which can be used at remote sites or at home to diagnose a range of indications, such as influenza. These indications can include influenza A, influenza B, streptococcal infections, or CT/NG infections. OTC products can include a consumable test card, wherein the test card is any of the assay formats (e.g., a lateral flow assay) disclosed herein. In an OTC product, the test card can be interpreted visually or using a mobile phone.
A number of support mediums are consistent with the devices, systems, fluidic devices, kits, and methods disclosed herein. These support mediums are, for example, consistent with fluidic devices disclosed herein for detection of a target nucleic acid (e.g., from an influenza virus) within the sample, wherein the fluidic device may comprise multiple pumps, valves, reservoirs, and chambers for sample preparation, amplification of a target nucleic acid within the sample, mixing with a programmable nuclease, and detection of a detectable signal arising from cleavage of detector nucleic acids by the programmable nuclease within the fluidic system itself. These support mediums are compatible with the samples, reagents, and fluidic devices described herein for detection of an ailment, such as a a viral infection, for example an infection from influenza A or influenza B. A support medium described herein can provide a way to present the results from the activity between the reagents and the sample. The support medium provides a medium to present the detectable signal in a detectable format. Optionally, the support medium concentrates the detectable signal to a detection spot in a detection region to increase the sensitivity, specificity, or accuracy of the assay. The support mediums can present the results of the assay and indicate the presence or absence of the disease of interest targeted by the target nucleic acid. The result on the support medium can be read by eye or using a machine. The support medium helps to stabilize the detectable signal generated by the cleaved detector molecule on the surface of the support medium. In some instances, the support medium is a lateral flow assay strip. In some instances, the support medium is a PCR plate. The PCR plate can have 96 wells or 384 wells. The PCR plate can have a subset number of wells of a 96 well plate or a 384 well plate. A subset number of wells of a 96 well PCR plate is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 wells. For example, a PCR subset plate can have 4 wells wherein a well is the size of a well from a 96 well PCR plate (e.g., a 4 well PCR subset plate wherein the wells are the size of a well from a 96 well PCR plate). A subset number of wells of a 384 well PCR plate is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, or 380 wells. For example, a PCR subset plate can have 20 wells wherein a well is the size of a well from a 384 well PCR plate (e.g., a 20 well PCR subset plate wherein the wells are the size of a well from a 384 well PCR plate). The PCR plate or PCR subset plate can be paired with a fluorescent light reader, a visible light reader, or other imaging device. Often, the imaging device is a digital camera, such a digital camera on a mobile device. The mobile device may have a software program or a mobile application that can capture an image of the PCR plate or PCR subset plate, identify the assay being performed, detect the individual wells and the sample therein, provide image properties of the individuals wells comprising the assayed sample, analyze the image properties of the contents of the individual wells, and provide a result.
The support medium has at least one specialized zone or region to present the detectable signal. The regions comprise at least one of a sample pad region, a nucleic acid amplification region, a conjugate pad region, a detection region, and a collection pad region. In some instances, the regions are overlapping completely, overlapping partially, or in series and in contact only at the edges of the regions, where the regions are in fluid communication with its adjacent regions. In some instances, the support medium has a sample pad located upstream of the other regions; a conjugate pad region having a means for specifically labeling the detector moiety; a detection region located downstream from sample pad; and at least one matrix which defines a flow path in fluid connection with the sample pad. In some instances, the support medium has an extended base layer on top of which the various zones or regions are placed. The extended base layer may provide a mechanical support for the zones.
Described herein are sample pad that provide an area to apply the sample to the support medium. The sample may be applied to the support medium by a dropper or a pipette on top of the sample pad, by pouring or dispensing the sample on top of the sample pad region, or by dipping the sample pad into a reagent chamber holding the sample. The sample can be applied to the sample pad prior to reaction with the reagents when the reagents are placed on the support medium or be reacted with the reagents prior to application on the sample pad. The sample pad region can transfer the reacted reagents and sample into the other zones of the support medium. Transfer of the reacted reagents and sample may be by capillary action, diffusion, convection or active transport aided by a pump. In some cases, the support medium is integrated with or overlayed by microfluidic channels to facilitate the fluid transport.
The dropper or the pipette may dispense a predetermined volume. In some cases, the predetermined volume may range from about 1 μl to about 1000 μl, about 1 μl to about 500 μl, about 1 μl to about 100 μl, or about 1 μl to about 50 μl. In some cases, the predetermined volume may be at least 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 25 μl, 50 μl, 75 μl, 100 μl, 250 μl, 500 μl, 750 μl, or 1000 μl. The predetermined volume may be no more than 5 μl, 10 μl, 25 μl, 50 μl, 75 μl, 100 μl, 250 μl, 500 μl, 750 μl, or 1000 μl. The dropper or the pipette may be disposable or be single-use.
Optionally, a buffer or a fluid may also be applied to the sample pad to help drive the movement of the sample along the support medium. In some cases, the volume of the buffer or the fluid may range from about 1 μl to about 1000 μl, about 1 μl to about 500 μl, about 1 μl to about 100 μl, or about 1 μl to about 50 μl. In some cases, the volume of the buffer or the fluid may be at least 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 25 μl, 50 μl, 75 μl, 100 μl, 250 μl, 500 μl, 750 μl, or 1000 μl. The volume of the buffer or the fluid may be no more than than 5 μl, 10 μl, 25 μl, 50 μl, 75 μl, 100 μl, 250 μl, 500 μl, 750 μl, or 1000 μl. In some cases, the buffer or fluid may have a ratio of the sample to the buffer or fluid of at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.
The sample pad can be made from various materials that transfer most of the applied reacted reagents and samples to the subsequent regions. The sample pad may comprise cellulose fiber filters, woven meshes, porous plastic membranes, glass fiber filters, aluminum oxide coated membranes, nitrocellulose, paper, polyester filter, or polymer-based matrices. The material for the sample pad region may be hydrophilic and have low non-specific binding. The material for the sample pad may range from about 50 μm to about 1000 μm, about 50 μm to about 750 μm, about 50 μm to about 500 μm, or about 100 μm to about 500 μm.
The sample pad can be treated with chemicals to improve the presentation of the reaction results on the support medium. The sample pad can be treated to enhance extraction of nucleic acid in the sample, to control the transport of the reacted reagents and sample or the conjugate to other regions of the support medium, or to enhance the binding of the cleaved detection moiety to the conjugate binding molecule on the surface of the conjugate or to the capture molecule in the detection region. The chemicals may comprise detergents, surfactants, buffers, salts, viscosity enhancers, or polypeptides. In some instances, the chemical comprises bovine serum albumin.
Described herein are conjugate pads that provide a region on the support medium comprising conjugates coated on its surface by conjugate binding molecules that can bind to the detector moiety from the cleaved detector molecule or to the control molecule. The conjugate pad can be made from various materials that facilitate binding of the conjugate binding molecule to the detection moiety from cleaved detector molecule and transfer of most of the conjugate-bound detection moiety to the subsequent regions. The conjugate pad may comprise the same material as the sample pad or other zones or a different material than the sample pad. The conjugate pad may comprise glass fiber filters, porous plastic membranes, aluminum oxide coated membranes, paper, cellulose fiber filters, woven meshes, polyester filter, or polymer-based matrices. The material for the conjugate pad region may be hydrophilic, have low non-specific binding, or have consistent fluid flow properties across the conjugate pad. In some cases, the material for the conjugate pad may range from about 50 μm to about 1000 μm, about 50 μm to about 750 μm, about 50 μm to about 500 μm, or about 100 μm to about 500 μm.
Further described herein are conjugates that are placed on the conjugate pad and immobilized to the conjugate pad until the sample is applied to the support medium. The conjugates may comprise a nanoparticle, a gold nanoparticle, a latex nanoparticle, a quantum dot, a chemiluminescent nanoparticle, a carbon nanoparticle, a selenium nanoparticle, a fluorescent nanoparticle, a liposome, or a dendrimer. The surface of the conjugate may be coated by a conjugate binding molecule that binds to the detection moiety from the cleaved detector molecule.
The conjugate binding molecules described herein coat the surface of the conjugates and can bind to detection moiety. The conjugate binding molecule binds selectively to the detection moiety cleaved from the detector nucleic acid. Some suitable conjugate binding molecules comprise an antibody, a polypeptide, or a single stranded nucleic acid. In some cases, the conjugate binding molecule binds a dye and a fluorophore. Some such conjugate binding molecules that bind to a dye or a fluorophore can quench their signal. In some cases, the conjugate binding molecule is a monoclonal antibody. In some cases, an antibody, also referred to as an immunoglobulin, includes any isotype, variable regions, constant regions, Fc region, Fab fragments, F(ab′)2 fragments, and Fab′ fragments. Alternatively, the conjugate binding molecule is a non-antibody compound that specifically binds the detection moiety. Sometimes, the conjugate binding molecule is a polypeptide that can bind to the detection moiety. Sometimes, the conjugate binding molecule is avidin or a polypeptide that binds biotin. Sometimes, the conjugate binding molecule is a detector moiety binding nucleic acid.
The diameter of the conjugate may be selected to provide a desired surface to volume ratio. In some instances, a high surface area to volume ratio may allow for more conjugate binding molecules that are available to bind to the detection moiety per total volume of the conjugates. In some cases, the diameter of the conjugate may range from about 1 nm to about 1000 nm, about 1 nm to about 500 nm, about 1 nm to about 100 nm, or about 1 nm to about 50 nm. In some cases, the diameter of the conjugate may be at least 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or 1000 nm. In some cases, the diameter of the conjugate may be no more than 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or 1000 nm.
The ratio of conjugate binding molecules to the conjugates can be tailored to achieve desired binding properties between the conjugate binding molecules and the detection moiety. In some instances, the molar ratio of conjugate binding molecules to the conjugates is at least 1:1, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, or 1:500. In some instances, the mass ratio of conjugate binding molecules to the conjugates is at least 1:1, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, or 1:500. In some instances, the number of conjugate binding molecules per conjugate is at least 1, 10, 50, 100, 500, 1000, 5000, or 10000.
The conjugate binding molecules can be bound to the conjugates by various approached. Sometimes, the conjugate binding molecule can be bound to the conjugate by passive binding. Some such passive binding comprise adsorption, absorption, hydrophobic interaction, electrostatic interaction, ionic binding, or surface interactions. In some cases, the conjugate binding molecule can be bound to the conjugate covalently. Sometimes, the covalent bonding of the conjugate binding molecule to the conjugate is facilitated by EDC/NHS chemistry or thiol chemistry.
Described herein are detection region on the support medium that provide a region for presenting the assay results. The detection region can be made from various materials that facilitate binding of the conjugate-bound detection moiety from cleaved detector molecule to the capture molecule specific for the detection moiety. The detection pad may comprise the same material as other zones or a different material than the other zones. The detection region may comprise nitrocellulose, paper, cellulose, cellulose fiber filters, glass fiber filters, porous plastic membranes, aluminum oxide coated membranes, woven meshes, polyester filter, or polymer-based matrices. Often the detection region may comprise nitrocellulose. The material for the region pad region may be hydrophilic, have low non-specific binding, or have consistent fluid flow properties across the region pad. The material for the conjugate pad may range from about 10 μm to about 1000 μm, about 10 μm to about 750 μm, about 10 μm to about 500 μm, or about 10 μm to about 300 μm.
The detection region comprises at least one capture area with a high density of a capture molecule that can bind to the detection moiety from cleaved detection molecule and at least one area with a high density of a positive control capture molecule. The capture area with a high density of capture molecule or a positive control capture molecule may be a line, a circle, an oval, a rectangle, a triangle, a plus sign, or any other shapes. In some instances, the detection region comprise more than one capture area with high densities of more than one capture molecules, where each capture area comprises one type of capture molecule that specifically binds to one type of detection moiety from cleaved detection molecule and are different from the capture molecules in the other capture areas. The capture areas with different capture molecules may be overlapping completely, overlapping partially, or spatially separate from each other. In some instances, the capture areas may overlap and produce a combined detectable signal distinct from the detectable signals generated by the individual capture areas. Usually, the positive control spot is spatially distinct from any of the detection spot.
The capture molecule described herein bind to detection moiety and immobilized in the detection spot in the detect region. Some suitable capture molecules comprise an antibody, a polypeptide, or a single stranded nucleic acid. In some cases, the capture molecule binds a dye and a fluorophore. Some such capture molecules that bind to a dye or a fluorophore can quench their signal. Sometimes, the capture molecule is an antibody that that binds to a dye or a fluorophore can quench their signal. In some cases, the capture molecule is a monoclonal antibody. In some cases, an antibody, also referred to as an immunoglobulin, includes any isotype, variable regions, constant regions, Fc region, Fab fragments, F(ab′)2 fragments, and Fab′ fragments. Alternatively, the capture molecule is a non-antibody compound that specifically binds the detection moiety. Sometimes, the capture molecule is a polypeptide that can bind to the detection moiety. In some instances, the detection moiety from cleaved detection molecule has a conjugate bound to the detection moiety, and the conjugate-detection moiety complex may bind to the capture molecule specific to the detection moiety on the detection region. Sometimes, the capture molecule is a polypeptide that can bind to the detection moiety. Sometimes, the capture molecule is avidin or a polypeptide that binds biotin. Sometimes, the capture molecule is a detector moiety binding nucleic acid.
The detection region described herein comprises at least one area with a high density of a positive control capture molecule. The positive control spot in the detection region provides a validation of the assay and a confirmation of completion of the assay. If the positive control spot is not detectable by the visualization methods described herein, the assay is not valid and should be performed again with a new system or kit. The positive control capture molecule binds at least one of the conjugate, the conjugate binding molecule, or detection moiety and is immobilized in the positive control spot in the detect region. Some suitable positive control capture molecules comprise an antibody, a polypeptide, or a single stranded nucleic acid. In some cases, the positive control capture molecule binds to the conjugate binding molecule. Some such positive control capture molecules that bind to a dye or a fluorophore can quench their signal. Sometimes, the positive control capture molecule is an antibody that that binds to a dye or a fluorophore can quench their signal. In some cases, the positive control capture molecule is a monoclonal antibody. In some cases, an antibody includes any isotype, variable regions, constant regions, Fc region, Fab fragments, F(ab′)2 fragments, and Fab′ fragments. Alternatively, the positive control capture molecule is a non-antibody compound that specifically binds the detection moiety. Sometimes, the positive control capture molecule is a polypeptide that can bind to at least one of the conjugate, the conjugate binding molecule, or detection moiety. In some instances, the conjugate unbound to the detection moiety binds to the positive control capture molecule specific to at least one of the conjugate, the conjugate binding molecule.
The kit or system described herein may also comprise a positive control sample to determine that the activity of at least one of programmable nuclease, a guide nucleic acid, or a single stranded detector nucleic acid. Often, the positive control sample comprises a target nucleic acid that binds to the guide nucleic acid. The positive control sample is contacted with the reagents in the same manner as the test sample and visualized using the support medium. The visualization of the positive control spot and the detection spot for the positive control sample provides a validation of the reagents and the assay.
The kit or system for detection of a target nucleic acid described herein further can comprises reagents protease treatment of the sample. The sample can be treated with protease, such as Protease K, before amplification or before assaying for a detectable signal. Often, a protease treatment is for no more than 15 minutes. Sometimes, the protease treatment is for no more than 1, 5, 10, 15, 20, 25, 30, or more minutes, or any value from 1 to 30 minutes.
The kit or system for detection of a target nucleic acid described herein further comprises reagents for nucleic acid amplification of target nucleic acids in the sample. Isothermal nucleic acid amplification allows the use of the kit or system in remote regions or low resource settings without specialized equipment for amplification. Often, the reagents for nucleic acid amplification comprise a recombinase, a oligonucleotide primer, a single-stranded DNA binding (SSB) protein, and a polymerase. Sometimes, nucleic acid amplification of the sample improves at least one of sensitivity, specificity, or accuracy of the assay in detecting the target nucleic acid. In some cases, the nucleic acid amplification is performed in a nucleic acid amplification region on the support medium. Alternatively or in combination, the nucleic acid amplification is performed in a reagent chamber, and the resulting sample is applied to the support medium. Sometimes, the nucleic acid amplification is isothermal nucleic acid amplification. In some cases, the nucleic acid amplification is transcription mediated amplification (TMA). Nucleic acid amplification is helicase dependent amplification (HDA) or circular helicase dependent amplification (cHDA) in other cases. In additional cases, nucleic acid amplification is strand displacement amplification (SDA). In some cases, nucleic acid amplification is by recombinase polymerase amplification (RPA). In some cases, nucleic acid amplification is by at least one of loop mediated amplification (LAMP) or the exponential amplification reaction (EXPAR). Nucleic acid amplification is, in some cases, by rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence based amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), or improved multiple displacement amplification (IMDA). Often, the nucleic acid amplification is performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes, or any value from 1 to 60 minutes. Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 20-45° C. In some cases, the nucleic acid amplification reaction is performed at a temperature no greater than 20° C., 25° C., 30° C., 35° C., 37° C., 40° C., 45° C., or any value from 20° C. to 45° C. In some cases, the nucleic acid amplification reaction is performed at a temperature of at least 20° C., 25° C., 30° C., 35° C., 37° C., 40° C., or 45° C., or any value from 20° C. to 45° C.
Sometimes, the total time for the performing the method described herein is no greater than 3 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, or any value from 3 hours to 20 minutes. Often, a method of nucleic acid detection from a raw sample comprises protease treating the sample for no more than 15 minutes, amplifying (can also be referred to as pre-amplyfing) the sample for no more than 15 minutes, subjecting the sample to a programmable nuclease-mediated detection, and assaying nuclease mediated detection. The total time for performing this method, sometimes, is no greater than 3 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, or any value from 3 hours to 20 minutes. Often, the protease treatment is Protease K. Often the amplifying is thermal cycling amplification. Sometimes the amplifying is isothermal amplification.
Described herein are collection pad region that provide a region to collect the sample that flows down the support medium. Often the collection pads are placed downstream of the detection region and comprise an absorbent material. The collection pad can increase the total volume of sample that enters the support medium by collecting and removing the sample from other regions of the support medium. This increased volume can be used to wash unbound conjugates away from the detection region to lower the background and enhance assay sensitivity. When the design of the support medium does not include a collection pad, the volume of sample analyzed in the support medium may be determined by the bed volume of the support medium. The collection pad may provide a reservoir for sample volume and may help to provide capillary force for the flow of the sample down the support medium.
The collection pad may be prepared from various materials that are highly absorbent and able to retain fluids. Often the collection pads comprise cellulose filters. In some instances, the collection pads comprise cellulose, cotton, woven meshes, polymer-based matrices. The dimension of the collection pad, usually the length of the collection pad, may be adjusted to change the overall volume absorbed by the support medium.
The support medium described herein may have a barrier around the edge of the support medium. Often the barrier is a hydrophobic barrier that facilitates the maintenance of the sample within the support medium or flow of the sample within the support medium. Usually, the transport rate of the sample in the hydrophobic barrier is much lower than through the regions of the support medium. In some cases, the hydrophobic barrier is prepared by contacting a hydrophobic material around the edge of the support medium. Sometimes, the hydrophobic barrier comprises at least one of wax, polydimethylsiloxane, rubber, or silicone.
Any of the regions on the support medium can be treated with chemicals to improve the visualization of the detection spot and positive control spot on the support medium. The regions can be treated to enhance extraction of nucleic acid in the sample, to control the transport of the reacted reagents and sample or the conjugate to other regions of the support medium, or to enhance the binding of the cleaved detection moiety to the conjugate binding molecule on the surface of the conjugate or to the capture molecule in the detection region. The chemicals may comprise detergents, surfactants, buffers, salts, viscosity enhancers, or polypeptides. In some instances, the chemical comprises bovine serum albumin. In some cases, the chemicals or physical agents enhance flow of the sample with a more even flow across the width of the region. In some cases, the chemicals or physical agents provide a more even mixing of the sample across the width of the region. In some cases, the chemicals or physical agents control flow rate to be faster or slower in order to improve performance of the assay. Sometimes, the performance of the assay is measured by at least one of shorter assay time, longer times during cleavage activity, longer or shorter binding time with the conjugate, sensitivity, specificity, or accuracy.
The devices, systems, fluidic devices, kits, and methods described herein can be multiplexed in a number of ways. These methods of multiplexing are, for example, consistent with fluidic devices disclosed herein for detection of a target nucleic acid within the sample, wherein the fluidic device may comprise multiple pumps, valves, reservoirs, and chambers for sample preparation, amplification of one or more than one sequences of target nucleic acids within the sample, mixing with a programmable nuclease, and detection of a detectable signal arising from cleavage of detector nucleic acids by the programmable nuclease within the fluidic system itself.
Methods consistent with the present disclosure include a multiplexing method of assaying for a target nucleic acid in a sample. A multiplexing method comprises contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; and assaying for a signal indicating cleavage of at least some protein-nucleic acids of a population of protein-nucleic acids, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. As another example, multiplexing method of assaying for a target nucleic acid in a sample, for example, comprises: a) contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; b) contacting the complex to a substrate; c) contacting the substrate to a reagent that differentially reacts with a cleaved substrate; and d) assaying for a signal indicating cleavage of the substrate, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. Often, the substrate is an enzyme-nucleic acid. Sometimes, the substrate is an enzyme substrate-nucleic acid.
Multiplexing can be either spatial multiplexing wherein multiple different target nucleic acids at the same time, but the reactions are spatially separated. Often, the multiple target nucleic acids are detected using the same programmable nuclease, but different guide nucleic acids. The multiple target nucleic acids sometimes are detected using the different programmable nucleases. Sometimes, multiplexing can be single reaction multiplexing wherein multiple different target acids are detected in a single reaction volume. Often, at least two different programmable nucleases are used in single reaction multiplexing. For example, multiplexing can be enabled by immobilization of multiple categories of detector nucleic acids within a fluidic system, to enable detection of multiple target nucleic acids within a single fluidic system. Multiplexing allows for detection of multiple target nucleic acids in one kit or system. In some cases, the multiple target nucleic acids comprise different target nucleic acids to a virus, such as an influenza virus. In some cases, the multiple target nucleic acids comprise different target nucleic acids associated withinfluenza and another disease (e.g., sepsis or a respiratory infection, such as an upper respiratory tract virus). Multiplexing for one disease increases at least one of sensitivity, specificity, or accuracy of the assay to detect the presence of the disease in the sample. In some cases, the multiple target nucleic acids comprise target nucleic acids directed to different viruses, bacteria, or pathogens responsible for more than one disease. In some cases, multiplexing allows for discrimination between multiple target nucleic acids, such as target nucleic acids that comprise different genotypes of the same bacteria or pathogen responsible for a disease, for example, for a wild-type genotype of a bacteria or pathogen and for genotype of a bacteria or pathogen comprising a mutation, such as a single nucleotide polymorphism (SNP) that can confer resistance to a treatment, such as antibiotic treatment. For example, multiplexing comprises method of assaying comprising a single assay for a microorganism species using a first programmable nuclease and an antibiotic resistance pattern in a microorganism using a second programmable nuclease. Sometimes, multiplexing allows for discrimination between multiple target nucleic acids of different influenza strains, for example, influenza A and influenza B. Often, multiplexing allows for discrimination between multiple target nucleic acids, such as target nucleic acids that comprise different genotypes, for example, for a wild-type genotype and for SNP genotype. Multiplexing for multiple viral infections provides the capability to test a panel of diseases from a single sample. For example, multiplexing for multiple diseases can be valuable in a broad panel testing of a new patient or in epidemiological surveys. Often multiplexing is used for identifying bacterial pathogens in sepsis or other diseases associated with multiple pathogens.
Furthermore, signals from multiplexing can be quantified. For example, a method of quantification for a disease panel comprises assaying for a plurality of unique target nucleic acids in a plurality of aliquots from a sample, assaying for a control nucleic acid control in a second aliquot of the sample, and quantifying a plurality of signals of the plurality of unique target nucleic acids by measuring signals produced by cleavage of detector nucleic acids compared to the signal produced in the second aliquot. Often the plurality of unique target nucleic acids are from a plurality of viruses in the sample. Sometimes the quantification of a signal of the plurality correlates with a concentration of a unique target nucleic acid of the plurality for the unique target nucleic acid of the plurality that produced the signal of the plurality. The disease panel can be for any disease, such as influenza.
The devices, systems, fluidic devices, kits, and methods described herein can be multiplexed by various configurations of the reagents and the support medium. In some cases, the kit or system is designed to have multiple support mediums encased in a single housing. Sometimes, the multiple support mediums housed in a single housing share a single sample pad. The single sample pad may be connected to the support mediums in various designs such as a branching or a radial formation. Alternatively, each of the multiple support mediums has its own sample pad. In some cases, the kit or system is designed to have a single support medium encased in a housing, where the support medium comprises multiple detection spots for detecting multiple target nucleic acids. Sometimes, the reagents for multiplexed assays comprise multiple guide nucleic acids, multiple programmable nucleases, and multiple single stranded detector nucleic acids, where a combination of one of the guide nucleic acids, one of the programmable nucleases, and one of the single stranded detector nucleic acids detects one target nucleic acid and can provide a detection spot on the detection region. In some cases, the combination of a guide nucleic acid, a programmable nuclease, and a single stranded detector nucleic acid configured to detect one target nucleic acid is mixed with at least one other combination in a single reagent chamber. In some cases, the combination of a guide nucleic acid, a programmable nuclease, and a single stranded detector nucleic acid configured to detect one target nucleic acid is mixed with at least one other combination on a single support medium. When these combinations of reagents are contacted with the sample, the reaction for the multiple target nucleic acids occurs simultaneously in the same medium or reagent chamber. Sometimes, this reacted sample is applied to the multiplexed support medium described herein.
In some cases, the combination of a guide nucleic acid, a programmable nuclease, and a single stranded detector nucleic acid configured to detect one target nucleic acid is provided in its own reagent chamber or its own support medium. In this case, multiple reagent chambers or support mediums are provided in the device, kit, or system, where one reagent chamber is designed to detect one target nucleic acid. In this case, multiple support mediums are used to detect the panel of viral infections, or other diseases of interest.
In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 2 different target nucleic acids in a single reaction. In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 3 different target nucleic acids in a single reaction. In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 4 different target nucleic acids in a single reaction. In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 5 different target nucleic acids in a single reaction. In some cases, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 6, 7, 8, 9, or 10 different target nucleic acids in a single reaction. In some instances, the multiplexed kits detect at least 2 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect at least 3 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect at least 4 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect at least 5 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect at least 6, 7, 8, 9, or 10 different target nucleic acids in a single kit.
A support medium as described herein can be housed in a number of ways that are consistent with the devices, systems, fluidic devices, kits, and methods disclosed herein. The housing for the support medium are, for example, consistent with fluidic devices disclosed herein for detection of a target nucleic acid within the sample, wherein the fluidic device may comprise multiple pumps, valves, reservoirs, and chambers for sample preparation, amplification of a target nucleic acid within the sample, mixing with a programmable nuclease, and detection of a detectable signal arising from cleavage of detector nucleic acids by the programmable nuclease within the fluidic system itself. For example, the fluidic device may be comprise support mediums to channel the flow of fluid from one chamber to another and wherein the entire fluidic device is encased within the housing described herein. Typically, the support medium described herein is encased in a housing to protect the support medium from contamination and from disassembly. The housing can be made of more than one part and assembled to encase the support medium. In some instances, a single housing can encase more than one support medium. The housing can be made from cardboard, plastics, polymers, or materials that provide mechanical protection for the support medium. Often, the material for the housing is inert or does not react with the support medium or the reagents placed on the support medium. The housing may have an upper part which when in place exposes the sample pad to receive the sample and has an opening or window above the detection region to allow the results of the lateral flow assay to be read. The housing may have guide pins on its inner surface that are placed around and on the support medium to help secure the compartments and the support medium in place within the housing. In some cases, the housing encases the entire support medium. Alternatively, the sample pad of the support medium is not encased and is left exposed to facilitate the receiving of the sample while the rest of the support medium is encased in the housing.
The housing and the support medium encased within the housing may be sized to be small, portable, and hand held. The small size of the housing and the support medium would facilitate the transport and use of the assay in remote regions or low resource settings. In some cases, the housing has a length of no more than 30 cm, 25 cm, 20 cm, 15 cm, 10 cm, or 5 cm. In some cases, the housing has a length of at least 1 cm, 5 cm, 10 cm, 15 cm, 20 cm, 25 cm, or 30 cm. In some cases, the housing has a width of no more than 30 cm, 25 cm, 20 cm, 15 cm, 10 cm, 5 cm, 4 cm, 3 cm, 2 cm, or 1 cm. In some cases, the housing has a width of at least 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 10 cm, 15 cm, 20 cm, 25 cm, or 30 cm. In some cases, the housing has a height of no more than 10 cm, 9 cm, 8 cm, 7 cm, 6 cm, 5 cm, 4 cm, 3 cm, 2 cm, or 1 cm. In some cases, the housing has a height of at least 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm. Typically, the housing is rectangular in shape.
The housing may comprise more than one piece. The housing may comprise an over-molding. The housing may seal a chamber, channel, compartment, or valve from the surrounding environment. The housing may be comprise sealable materials, such as polycarbonate capable of laser bonding. The housing may comprise a rigid material. The housing may comprise a flexible material. The housing may comprise connectors or adaptors. A set of connectors or adaptors may have tight tolerances. A set of connectors or adaptors may have loose tolerances.
In some instances, the housing provides additional information on the outer surface of the upper cover to facilitate the identification of the test type, visualization of the detection region, and analysis of the results. The upper outer housing may have identification label including but not limited to barcodes, QR codes, identification label, or other visually identifiable labels. In some instances, the identification label is imaged by a camera on a mobile device, and the image is analyzed to identify the disease that is being tested for. The correct identification of the test is important to accurately visualize and analyze the results. In some instances, the upper outer housing has fiduciary markers to orient the detection region to distinguish the positive control spot from the detection spots. In some instances, the upper outer housing has a color reference guide. When the detection region is imaged with the color reference guide, the detection spots, located using the fiduciary marker, can be compared with the positive control spot and the color reference guide to determine various image properties of the detection spot such as color, color intensity, and size of the spot. In some instances, the color reference guide has red, green, blue, black, and white colors. In some cases, the image of the detection spot can be normalized to at least one of the reference colors of the color reference guide, compared to at least two of the reference colors of the color reference guide, and generate a value for the detection spot. Sometimes, the comparison to at least two of the reference colors is comparison to a standard reference scale. In some instance, the image of the detection spot in some instance undergoes transformation or filtering prior to analysis. Analysis of the image properties of the detection spot can provide information regarding presence or absence of the target nucleic acid targeted by the assay and the disease associated with the target nucleic acid. In some instances, the analysis provides a qualitative result of presence or absence of the target nucleic acid in the sample. In some instances, the analysis provides a semi-quantitative or quantitative result of the level of the target nucleic acid present in the sample. Quantification may be performed by having a set of standards in spots/wells and comparing the test sample to the range of standards. A more semi-quantitative approach may be performed by calculating the color intensity of 2 spots/well compared to each other and measuring if one spot/well is more intense than the other. Sometimes, quantification is of quantification of circulating nucleic acid. The circulating nucleic acid can comprise a target nucleic acid. For example, a method of circulating nucleic acid quantification comprises assaying for a target nucleic acid of circulating nucleic acid in a first aliquot of a sample, assaying for a control nucleic acid in a second aliquot of the sample, and quantifying the target nucleic acid target in the first aliquot by measuring a signal produced by cleavage of a detector nucleic acid. Sometimes, a method of circulating RNA quantification comprises assaying for a target nucleic acid of the circulating RNA in a first aliquot of a sample, assaying for a control nucleic acid in a second aliquot of the sample, and quantifying the target nucleic acid target in the first aliquot by measuring a signal produced by cleavage of a detector nucleic acid. Often, the output comprises fluorescence/second. The reaction rate, sometimes, is log linear for output signal and target nucleic acid concentration. In some instances, the signal output is correlated with the target nucleic acid concentration. Sometimes, the circulating nucleic acid is DNA.
A number of detection or visualization devices and methods are consistent with the devices, systems, fluidic devices, kits, and methods disclosed herein. Methods of detection/visualization are, for example, consistent with fluidic devices disclosed herein for detection of a target nucleic acid within the sample, wherein the fluidic device may comprise multiple pumps, valves, reservoirs, and chambers for sample preparation, amplification of a target nucleic acid within the sample, mixing with a programmable nuclease, and detection of a detectable signal arising from cleavage of detector nucleic acids by the programmable nuclease within the fluidic system itself. For example, the fluidic device may comprise an incubation and detection chamber or a stand-alone detection chamber, in which a colorimetric, fluorescence, electrochemical, or electrochemiluminesence signal is generated for detection/visualization. Sometimes, the signal generated for detection is a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. Often a calorimetric signal is heat produced after cleavage of the detector nucleic acids. Sometimes, a calorimetric signal is heat absorbed after cleavage of the detector nucleic acids. A potentiometric signal, for example, is electrical potential produced after cleavage of the detector nucleic acids. An amperometric signal can be movement of electrons produced after the cleavage of detector nucleic acid. Often, the signal is an optical signal, such as a colorimetric signal or a fluorescence signal. An optical signal is, for example, a light output produced after the cleavage of the detector nucleic acids. Sometimes, an optical signal is a change in light absorbance between before and after the cleavage of detector nucleic acids. Often, a piezo-electric signal is a change in mass between before and after the cleavage of the detector nucleic acid. Sometimes, the detector nucleic acid is protein-nucleic acid. Often, the protein-nucleic acid is an enzyme-nucleic acid. The detection/visualization can be analyzed using various methods, as further described below. The results from the detection region from a completed assay can be visualized and analyzed in various ways. In some cases, the positive control spot and the detection spot in the detection region is visible by eye, and the results can be read by the user. In some cases, the positive control spot and the detection spot in the detection region is visualized by an imaging device. Often, the imaging device is a digital camera, such a digital camera on a mobile device. The mobile device may have a software program or a mobile application that can capture an image of the support medium, identify the assay being performed, detect the detection region and the detection spot, provide image properties of the detection spot, analyze the image properties of the detection spot, and provide a result. Alternatively or in combination, the imaging device can capture fluorescence, ultraviolet (UV), infrared (IR), or visible wavelength signals. The imaging device may have an excitation source to provide the excitation energy and captures the emitted signals. In some cases, the excitation source can be a camera flash and optionally a filter. In some cases, the imaging device is used together with an imaging box that is placed over the support medium to create a dark room to improve imaging. The imaging box can be a cardboard box that the imaging device can fit into before imaging. In some instances, the imaging box has optical lenses, mirrors, filters, or other optical elements to aid in generating a more focused excitation signal or to capture a more focused emission signal. Often, the imaging box and the imaging device are small, handheld, and portable to facilitate the transport and use of the assay in remote or low resource settings.
In some cases, detection or visualization may comprise the production of light by a diode. In some cases, a diode may produce visible light. In some cases, a diode may produce infrared light. In some cases, a diode may produce ultraviolet light. In some cases, a diode may be capable of producing different wavelengths or spectra of light. A diode may produce light over a broad or narrow spectrum. A diode may produce white light covering a large portion of the visible spectrum. A diode may produce a specific wavelength of light (e.g., a roughly Gaussian or Lorentzian wavelength vs intensity profile centered around a particular wavelength). In some cases, the bandwidth of light produced by a diode may be defined as the full width at half maximum intensity of a Gaussian-like or Lorentzian-like band. Some diodes produce light with narrow emission bandwidths. A diode may produce light with less than a 1 nm bandwidth. A diode may produce light with less than a 5 nm bandwidth. A diode may produce light with less than a 10 nm bandwidth. A diode may produce light with less than a 20 nm bandwidth. A diode may produce light with less than a 30 nm bandwidth. A diode may produce light with less than a 50 nm bandwidth. A diode may produce light with less than a 100 nm bandwidth. A diode may produce light with less than a 150 nm bandwidth. A diode may produce light with less than a 200 nm bandwidth.
In some cases, detection or visualization may comprise light detection by a diode (e.g., a photodiode). The current produced by a diode may be used to determine characteristics of light absorbed, including polarization, wavelength, intensity, direction traveled, point of origin, or any combination thereof. In some cases, detection or visualization may comprise light detection by a camera (e.g., a charge coupled device (CCD) detector) or a metal—oxide—semiconductor (MOS) detector). A detector (e.g., a photodiode, a CCD detector, or a MOS detector) may be configured to detect a bandwidth of light. In some cases, the bandwidth of light detected by a detector may be defined as the full width at half maximum intensity of a Gaussian-like or Lorentzian-like band. In some cases, the bandwidth of light detected by a detector may be narrowed by an emission filter positioned between the sample and the detector. The emission filter may be a long pass filter. The emission filter may be bandpass filter. The emission filter may be a notch filter. In some embodiments, the bandwidth of light detected by the detector may be less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 75 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, less than about 10 nm, or less than about 5 nm.
In some cases, a diode array may be used to excite and detect fluorescence from a sample. In some cases, a device may comprise a light producing diode and detector diode positioned to illuminate and detect light from a particular portion of a sample. In some cases, a device may comprise a light producing diode and detector diode positioned to illuminate and detect light from a particular sample compartment or chamber.
The assay described herein can be visualized and analyzed by a mobile application (app) or a software program. Using the graphic user interface (GUI) of the app or program, an individual can take an image of the support medium, including the detection region, barcode, reference color scale, and fiduciary markers on the housing, using a camera on a mobile device. The program or app reads the barcode or identifiable label for the test type, locate the fiduciary marker to orient the sample, and read the detectable signals, compare against the reference color grid, and determine the presence or absence of the target nucleic acid, which indicates the presence of the gene, virus, or the agent responsible for the disease. The mobile application can present the results of the test to the individual. The mobile application can store the test results in the mobile application. The mobile application can communicate with a remote device and transfer the data of the test results. The test results can be viewable remotely from the remote device by another individual, including a healthcare professional. A remote user can access the results and use the information to recommend action for treatment, intervention, clean up of an environment.
The support medium may be assembled with a variety of materials and reagents. Reagents may be dispensed or coated on to the surface of the material for the support medium. The material for the support medium may be laminated to a backing card, and the backing card may be singulated or cut into individual test strips. The device may be manufactured by completely manual, batch-style processing; or a completely automated, in-line continuous process; or a hybrid of the two processing approaches. The batch process may start with sheets or rolls of each material for the support medium. Individual zones of the support medium may be processed independently for dispensing and drying, and the final support medium may be assembled with the independently prepared zones and cut. The batch processing scheme may have a lower cost of equipment, and a higher labor cost than more automated in-line processing, which may have higher equipment costs. In some instances, batch processing may be preferred for low volume production due to the reduced capital investment. In some instances, automated in-line processing may be preferred for high volume production due to reduced production time. Both approaches may be scalable to production level.
In some instances, the support mediums are prepared using various instruments, including an XYZ-direction motion system with dispensers, impregnation tanks, drying ovens, a manual or semi-automated laminator, and cutting methods for reducing roll or sheet stock to appropriate lengths and widths for lamination. For dispensing the conjugate binding molecules for the conjugate zone and capture molecules for the detection zones, an XYZ-direction motion system with dispensers may be used. In some embodiments, the dispenser may dispense by a contact method or a non-contact method.
In automated or semi-automated preparation of the support medium, the support medium may be prepared from rolls of membranes for each region that are ordered into the final assembled order and unfurled from the rolls. For example, the membranes can be ordered from sample pad region to collection pad region from left to right with one membrane corresponding to a region on the support medium, all onto an adhesive cardstock. The dispenser places the reagents, conjugates, detection molecules, and other treatments for the membrane onto the membrane. The dispensed fluids are dried onto the membranes by heat, in a low humidity chamber, or by freeze drying to stabilize the dispensed molecules. The membranes are cut into strips and placed into the housing and packaged.
Disclosed herein are various fluidic devices for detection of a target nucleic acid of interest in a biological sample. The fluidic devices described in detail below can be used to monitor the reaction of target nucleic acids in samples with a programmable nuclease, thereby allowing for the detection of said target nucleic acid. All samples and reagents disclosed herein are compatible for use with a fluidic device disclosed below. Any programmable nuclease, such as any Cas nuclease described herein, are compatible for use with a fluidic device disclosed below. Support mediums and housing disclosed herein are also compatible for use in conjunction with the fluidic devices disclosed below. Multiplexing detection, as described throughout the present disclosure, can be carried out within the fluidic devices disclosed herein. Compositions and methods for detection and visualization disclosed herein are also compatible for use within the below described fluidic systems.
In the below described fluidic systems, any programmable nuclease (e.g., CRISPR-Cas) reaction can be monitored. For example, any programmable nuclease disclosed herein can be used to cleave the reporter molecules to generate a detection signal. In some cases, the programmable nuclease is Cas13. Sometimes the Cas13 is Cas13a, Cas13b, Cas13c, Cas13d, or Cas13e. In some cases, the programmable nuclease is Mad7 or Mad2. In some cases, the programmable nuclease is Cas12. Sometimes the Cas12 is Cas12a, Cas12b, Cas12c, Cas12d, or Cas12e. In some cases, the programmable nuclease is Csm1, Cas9, C2c4, C2c8, C2c5, C2c10, C2c9, or CasZ. Sometimes, the Csm1 is also called smCms1, miCms1, obCms1, or suCms1. Sometimes Cas13a is also called C2c2. Sometimes CasZ is also called Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, or Cas14h. Sometimes, the programmable nuclease is a type V CRISPR-Cas system. In some cases, the programmable nuclease is a type VI CRISPR-Cas system. Sometimes the programmable nuclease is a type III CRISPR-Cas system. In some cases, the programmable nuclease is from at least one of Leptotrichia shahii (Lsh), Listeria seeligeri (Lse), Leptotrichia buccalis (Lbu), Leptotrichia wadeu (Lwa), Rhodobacter capsulatus (Rca), Herbinix hemicellulosilytica (Hhe), Paludibacter propionicigenes (Ppr), Lachnospiraceae bacterium (Lba), [Eubacterium] rectale (Ere), Listeria newyorkensis (Lny), Clostridium aminophilum (Cam), Prevotella sp. (Psm), Capnocytophaga canimorsus (Cca, Lachnospiraceae bacterium (Lba), Bergeyella zoohelcum (Bzo), Prevotella intermedia (Pin), Prevotella buccae (Pbu), Alistipes sp. (Asp), Riemerella anatipestifer (Ran), Prevotella aurantiaca (Pau), Prevotella saccharolytica (Psa), Prevotella intermedia (Pint), Capnocytophaga canimorsus (Cca), Porphyromonas gulae (Pgu), Prevotella sp. (Psp), Porphyromonas gingivalis (Pig), Prevotella intermedia (Pin3), Enterococcus italicus (Ei), Lactobacillus salivarius (Ls), or Thermus thermophilus (Tt). Sometimes the Cas13 is at least one of LbuCas13a, LwaCas13a, LbaCas13a, HheCas13a, PprCas13a, EreCas13a, CamCas13a, or LshCas13a.
A workflow of a method for detecting a target nucleic acid in a sample within a fluidic device can include sample preparation, nucleic acid amplification, incubation with a programmable nuclease, and/or detection (readout).
Workflows and systems compatible with the compositions and methods provided herein include one-pot reactions and two-pot reactions. In a one-pot reaction, amplification, reverse transcription, amplification and reverse transcription, or amplification and in vitro transcription, and detection can be carried out simultaneously in one chamber. In other words, in a one-pot reaction, any combination of reverse transcription, amplification, and in vitro transcription can be performed in the same reaction as detection. In a two-pot reaction, any combination of reverse transcription, amplification, and in vitro transcription can be performed in a first reaction, followed by detection in a second reaction. The one-pot or two-pot reactions can be carried out in any of the chambers of the devices disclosed herein.
A fluidic device for sample preparation can be referred to as a filtration device. In some embodiments, the filtration device for sample preparation resembles a syringe or, comprises, similar functional elements to a syringe. For example, a functional element of the filtration device for sample preparation includes a narrow tip for collection of liquid samples. Liquid samples can include blood, saliva, urine, or any other biological fluid. Liquid samples can also include liquid tissue homogenates. The tip, for collection of liquid samples, can be manufactured from glass, metal, plastic, or other biocompatible materials. The tip may be replaced with a glass capillary that may serve as a metering apparatus for the amount of biological sample added downstream to the fluidic device. For some samples, e.g., blood, the capillary may be the only fluidic device required for sample preparation. Another functional element of the filtration device for sample preparation may include a channel that can carry volumes from nL to mL, containing lysis buffers compatible with the programmable nuclease reaction downstream of this process. The channel may be manufactured from metal, plastic, or other biocompatible materials. The channel may be large enough to hold an entire fecal, buccal, or other biological sample collection swab. The filtration device may further contain a solution of reagents that will lyse the cells in each type of samples and release the nucleic acids so that they are accessible to the programmable nuclease. Active ingredients of the solution may be chaotropic agents, detergents, salts, and can be of high osmolality, ionic strength and pH. Chaotropic agents or chaotropes are substances that disrupt the three-dimensional structure in macromolecules such as proteins, DNA, or RNA. One example protocol comprises a 4 M guanidinium isothiocyanate, 25 mM sodium citrate. 2H2O, 0.5% (w/v) sodium lauryl sarcosinate, and 0.1 M β-mercaptoethanol), but numerous commercial buffers for different cellular targets may also be used. Alkaline buffers may also be used for cells with hard shells, particularly for environmental samples. Detergents such as sodium dodecyl sulphate (SDS) and cetyl trimethylammonium bromide (CTAB) may also be implemented to chemical lysis buffers. Cell lysis may also be performed by physical, mechanical, thermal or enzymatic means, in addition to chemically-induced cell lysis mentioned previously. The device may include more complex architecture depending on the type of sample, such as nanoscale barbs, nanowires, sonication capability in a separate chamber of the device, integrated laser, integrated heater, for example, a Peltier-type heater, or a thin-film planar heater, and/or microcapillary probes for electrical lysis. Any samples described herein can be used in this workflow. For example samples may include liquid samples collected from a subject being tested for a condition of interest.
A fluidic device may be used to carry out any one of, or any combination of, Steps 2-4 of
A fluidic device may comprise a plurality of chambers and types of chambers. A fluidic device may comprise a plurality of chambers configured to contain a sample with reagents and in conditions conducive to a particular type of reaction. Such a chamber may be designed to facilitate detection of a reaction or a reaction species (e.g., by having transparent surfaces so that the contents of the chamber can be monitored by an external fluorimeter, or by having electrodes capable of potentiometric analysis). A fluidic device may comprise an amplification chamber, which can be designed to contain a sample and reagents in conditions (e.g., temperature) suitable for an amplification reaction. A fluidic device may comprise a detection chamber, which may be designed to contain a sample with reagents in conditions suitable for a detection reaction (e.g., a colorimetric reaction or a DETECTR reaction). A fluidic device may also comprise chambers designed to store or transfer reagents. For example, a fluidic device may comprise an amplification reagent chamber designed to hold reagents for an amplification reaction (e.g., LAMP) or a detection reagent chamber designed to hold reagents for a reaction capable of detecting the presence or absence of a species (e.g., a DETECTR reaction). A fluidic device may comprise a chamber configured for multiple purposes (e.g., a chamber may be configured for storing a reagent, containing two types of samples for two separate types of reactions, and facilitating fluorescence detection).
A fluidic device may comprise a sample inlet (the term ‘sample inlet’ is herein used interchangeably with sample inlet port and sample collection port) that leads to an internal space within the fluidic device, such as a chamber or fluidic channel. A sample inlet may lead to a chamber within the fluidic device. A sample inlet may be capable of sealing. A sample inlet may be sealed such that fluid is prevented from passing through the sample inlet. In some cases, a sample inlet seals around a second apparatus designed to deliver a sample, thus sealing the sample inlet from the surrounding environment. For example, a sample inlet may be capable of sealing around a swab or syringe. A sample inlet may also be configured to accommodate a cap or other mechanism that covers or seals the A sample inlet may comprise a bendable or breakable component. For example, a sample inlet may comprise a seal that breaks upon sample insertion. In some cases, a seal within a sample inlet releases reagents upon breaking. A sample inlet may comprise multiple chambers or compartments. For example, a sample inlet may comprise an upper compartment and a lower compartment separated by a breakable plastic seal. The seal may break upon sample insertion, releasing contents (e.g., lysis buffer or amplification buffer) from the upper container into the lower container, where it may mix with the sample and elute into a separate compartment (e.g., a sample compartment) within the fluidic device.
In some embodiments, the fluidic device may be a pneumatic device. The pneumatic device may comprise one or more sample chambers connected to one or more detection chambers by one or more pneumatic valves. Optionally, the pneumatic device may further comprise one or more amplification chamber between the one or more sample chambers and the one or more detection chambers. The one or more amplification chambers may be connected to the one or more sample chambers and the one or more detection chambers by one or more pneumatic valves. A pneumatic valve may be made from PDMS, or any other suitable material. A pneumatic valve may comprise a channel perpendicular to a microfluidic channel connecting the chambers and allowing fluid to pass between chambers when the valve is open. In some embodiments, the channel deflects downward upon application of air pressure through the channel perpendicular to the microfluidic channel. In some embodiments, the fluidic device may be a sliding valve device. The sliding valve device may comprise a sliding layer with one or more channels and a fixed layer with one or more sample chambers and one or more detection chambers. Optionally, the fixed layer may further comprise one or more amplification chambers. In some embodiments, the sliding layer is the upper layer and the fixed layer is the lower layer. In other embodiments, the sliding layer is the lower layer and the fixed layer is the upper layer. The sliding valve device may further comprise one or more of a side channel with an opening aligned with an opening in the sample chamber, a side channel with an opening aligned with an opening in the amplification chamber, or a side channel with an opening aligned with the opening in the detection chamber. In some embodiments the side channels are connected to a mixing chamber to allow transfer of fluid between the chambers. In some embodiments, the sliding valve device comprises a pneumatic pump for mixing, aspirating, and dispensing fluid in the device.
In some embodiments, a fluidic device may comprise a sliding valve. A sliding valve may be capable of adopting multiple positions, that connect different channels or compartments in a device. In some cases, a sliding device comprises multiple sets of channels that can simultaneously connect multiple different channels or compartments. For example a device that comprises 10 amplification chambers, 10 reagent chambers, and 1 sample chamber may comprise a sliding valve that can adopt a first position connecting the sample chamber to the 10 amplification chambers through 10 separate channels, and a second position that may separately connect the 10 amplification chambers to the 10 reagent chambers. A sliding valve may be capable of automated control by a device or computer. A sliding valve may comprise a transfer fluidic channel, which can have a first end that is open to a first chamber or fluidic channel and a second end that is blocked when the sliding valve is in a first position, and can have the first end blocked and the second end open to a second chamber or fluidic channel when the sliding valve is in a second position. A sliding valve may be designed to combine the flow from two or more chambers or channels into a single chamber or channel. A sliding valve may be designed to divide the flow from a single chamber or channel into two or more separate chambers or fluidic channels.
The chip (also referred to as fluidic device) may be manufactured from a variety of different materials. Exemplary materials that may be used include plastic polymers, such as poly-methacrylate (PMMA), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene (PE), high-density polyethylene (HDPE), polypropylene (PP); glass; and silicon. Features of the chip may be manufactured by various processes. For example, features may be (1) embossed using injection molding, (2) micro-milled or micro-engraved using computer numerical control (CNC) micromachining, or non-contact laser drilling (by means of a CO2 laser source); (3) additive manufacturing, and/or (4) photolithographic methods. A chip may comprise a material or combination of materials that thermally isolate different portions of the chip (e.g., two fluidic channels or reaction chambers may be thermally isolated by intervening material between them).
A design may include a plurality of input ports operated by a plurality of pumps. For example, the design may include up to three (3) input ports operated by three (3) pumps, labelled on
The ports may be connected to pneumatic pressure pumps, air or gas may be pumped into the microfluidic channels to control the injection of fluids into the fluidic device. At least three reservoirs may be connected to the device, each containing buffered solutions of: (1) sample, which may be a solution containing purified nucleic acids processed in a separate fluidic device, or neat sample (blood, saliva, urine, stool, and/or sputum); (2) amplification mastermix, which varies depending on the method used, wherein the method may include any of loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), multiple displacement amplification (MDA), rolling circle amplification (RCA), and nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), circular helicase dependent amplification (cHDA), exponential amplification reaction (EXPAR), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), or improved multiple displacement amplification (IMDA); and (3) pre-complexed programmable nuclease mix, which includes one or more programmable nuclease and guide oligonucleotides. The method of nucleic acid amplification may also be polymerase chain reaction (PCR), which includes cycling of the incubation temperature at different levels, hence is not defined as isothermal. Often, the reagents for nucleic acid amplification comprise a recombinase, a oligonucleotide primer, a single-stranded DNA binding (SSB) protein, and a polymerase. Sometimes, nucleic acid amplification of the sample improves at least one of sensitivity, specificity, or accuracy of the assay in detecting the target nucleic acid. In some cases, the nucleic acid amplification is performed in a nucleic acid amplification region on the support medium. Alternatively or in combination, the nucleic acid amplification is performed in a reagent chamber, and the resulting sample is applied to the support medium. Sometimes, the nucleic acid amplification is isothermal nucleic acid amplification. Complex formation of a nuclease with guides (a programmable nuclease) and reporter probes may occur off the chip. An additional port for output of the final reaction products is depicted at the end of the fluidic path, and is operated by a similar pump, as the ones described for P1-P3. The reactions product can be, thus, collected for additional processing and/or characterization, e.g., sequencing.
A device may comprise a plurality of chambers, fluidic channels and valves. A device may comprise multiple types of chambers, fluidic channels, valves, or any combination thereof. A device may comprise different numbers of chambers, fluidic channels, and valves. For example, a device may comprise one sample chamber, a rotating valve connecting the sample chamber to 10 separate amplification reaction chambers, and two sliding valves controlling flow from the 10 amplification reaction chambers into 30 separate Detection chambers. A rotating valve may connect 2 or more chambers or fluidic channels. A rotating valve may connect 3 or more chambers or fluidic channels. A rotating valve may connect 4 or more chambers or fluidic channels. A rotating valve may connect 5 or more chambers or fluidic channels. A rotating valve may connect 8 or more chambers or fluidic channels. A rotating valve may connect 10 or more chambers or fluidic channels. A rotating valve may connect 15 or more chambers or fluidic channels. A rotating valve may connect 20 or more chambers or fluidic channels.
A fluidic device may comprise a plurality of channels. A fluidic device may comprise a plurality of channels comprising a plurality of dimensions and properties. A fluidic device may comprise two channels with identical lengths. A fluidic device may comprise two channels that provide identical resistance. A fluidic device may comprise two identical channels.
A fluidic device may comprise a millichannel. A millichannel may have a width of between 100 and 200 mm. A millichannel may have a width of between 50 and 100 nm. A millichannel may have a width of between 20 and 50 nm. A millichannel may have a width of between 10 and 20 nm. A millichannel may have a width of between 1 and 10 nm. A fluidic device may comprise a microchannel. A microchannel may have a width of between 800 and 990 μm. A microchannel may have a width of between 600 and 800 μm. A microchannel may have a width of between 400 and 600 μm. A microchannel may have a width of between 200 and 400 μm. A microchannel may have a width of between 100 and 200 μm. A microchannel may have a width of between 50 and 100 μm. A microchannel may have a width of between 30 and 50 μm. A microchannel may have a width of between 20 and 30 μm. A microchannel may have a width of between 10 and 20 μm. A microchannel may have a width of between 5 and 10 μm. A microchannel may have a width of between 1 and 5μm. A fluidic device may comprise a nanochannel. A nanochannel may have a width of between 800 and 990 nm. A nanochannel may have a width of between 600 and 800 nm. A nanochannel may have a width of between 400 and 600 nm. A nanochannel may have a width of between 200 and 400 nm. A nanochannel may have a width of between 1 and 200 nm. A channel may have a comparable height and width. A channel may have a greater width than height, or a narrower width than height. A channel may have a width that is 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000 or more times its height. A channel may have a width that is 0.9, 0.8, 0.7, 0.6, 0.5, 0.25, 0.1, 0.05, 0.01, 0.005, 0.001 times its height. A channel may have a width that is less than 0.001 times its height. A channel may have non-uniform dimensions. A channel may have different dimensions at different points along its length. A channel may divide into 2 or more separate channels. A channel may be straight, or may have bends, curves, turns, angles, or other features of non-linear shapes. A channel may comprise a loop or multiple loops.
A fluidic device may comprise a resistance channel. A resistance channel may be a channel with slow flow rates relative to other channels within the fluidic device. A resistance channel may be a channel with low volumetric flow rates relative to other channels within the fluidic device. A resistance channel may provide greater resistance to sample flow relative to other channels in the fluidic device. A resistance channel may prevent or limit sample backflow. A resistance channel may prevent or limit cross-contamination between multiple samples within a device by limiting turbulence. A resistance channel may contribute to flow stability within a fluidic device. A resistance channel may limit disparities in flow rates between multiple portions of a fluidic device. A resistance channel may stabilize flow rates within a device, and minimize flow variation over time.
The flow of liquid in a fluidic device may be controlled with a plurality of microvalves. For example, the flow of liquid in this fluidic device may be controlled using up to four (4) microvalves, labelled in
The flow to and from the fluidic channel from each of P1-P4 is controlled by valves, labelled as V1-V4. The volume of liquids pumped into the ports can vary from nL to mL depending in the overall size of the device.
In device iteration 2.1, shows in
In device iteration 2.2, shown in
In device iteration 2.3, shown in
During the detection step (shown as step 4 in the workflow diagram of
The fluorescence detection and excitation may be multiplexed, wherein, for example, fluorescence detection involves exciting and detecting more than one fluorophore in the incubation and detection chamber (C1 or C2). The fluorimeter itself may be multichannel, in which detecting and exciting light at different wavelengths, or more than one fluorimeter may be used in tandem, and their position above the incubation and detection chamber (C1 and C2) be modified by mechanical means, such as a motorized mechanism using micro or macro controllers and actuators (electric, electronic, and/or piezo-electric).
Two electrochemical detection variations are described herein, using integrated working, counter and reference electrodes in the incubation and detection chamber (C1 or C2):
Increase in signal. The progress of the cleavage reaction catalyzed by the programmable nuclease may be detected using a streptavidin-biotin coupled reaction. The top surface of the detection and incubation chamber may be functionalized with nucleic acid molecules (ssRNA, ssDNA or ssRNA/DNA hybrid molecules) conjugated with a biotin moiety. The bottom surface of the detection and incubation chamber operates as an electrode, comprising of working, reference, and counter areas, manufactured (or screen-printed) from carbon, graphene, silver, gold, platinum, boron-doped diamond, copper, bismuth, titanium, antimony, chromium, nickel, tin, aluminum, molybdenum, lead, tantalum, tungsten, steel, carbon steel, cobalt, indium tin oxide (ITO), ruthenium oxide, palladium, silver-coated copper, carbon nano-tubes, or other metals. The bottom surface of the detection and incubation chamber may be coated with streptavidin molecules. In the absence of any biotin molecules, the current measured by a connected electrochemical analyzer (commercial, or custom-made) is low. When the pre-complexed programmable nuclease mix with amplified target flows in the detection and incubation chamber, and is activated at a higher temperature, for example at 37° C., cleavage of the single-stranded nucleic acid (ssNA) linker releases biotin molecules that can diffuse onto the streptavidin-coated bottom surface of the detection and incubation chamber. Because of the interaction of biotin and streptavidin molecules, an increase in the current is read by a coupled electrochemical analyzer.
In some cases, reporter cleavage may increase the intensity of an electrochemical signal (e.g., a potentiometric signal from a square wave or cyclic voltammogram). Reporter cleavage may increase the diffusion constant of an electroactive moiety in the reporter, which can lead to an increase of an electrochemical signal. Thus, in some cases, electrochemical signal increase proportional to the degree of transcollateral reporter cleavage.
Some DETECTR experiments may be sensitive to small changes in cleaved reporter concentration, allowing low concentrations of target nucleic acid to be detected or distinguished. An electrochemical DETECTR assay (a DETECTR assay that utilizes electrochemical detection) may be capable to detecting less than 100 nM target nucleic acid. An electrochemical DETECTR assay may be capable to detecting less than 10 nM target nucleic acid. An electrochemical DETECTR assay may be capable to detecting less than 1 nM target nucleic acid. An electrochemical DETECTR assay may be capable to detecting less than 100 pM target nucleic acid. An electrochemical DETECTR assay may be capable to detecting less than 10 pM target nucleic acid. An electrochemical DETECTR assay may be capable to detecting less than 1 pM target nucleic acid. An electrochemical DETECTR assay may be capable to detecting less than 100 fM target nucleic acid. An electrochemical DETECTR assay may be capable to detecting less than 50 fM target nucleic acid. An electrochemical DETECTR assay may be capable to detecting less than 10 fM target nucleic acid. An electrochemical DETECTR assay may be capable to detecting less than 1 fM target nucleic acid. In some cases, an electrochemical detection may be more sensitive than fluorescence detection. In some cases, a DETECTR assay with electrochemical detection may have a lower detection limit than a DETECTR assay that utilizes fluorescence detection.
In some cases, an electrochemical DETECTR reaction may require low reporter concentrations. In some cases, an electrochemical DETECTR reaction may require low reporter concentrations. An electrochemical DETECTR reaction may require less than 10 μM reporter. An electrochemical DETECTR reaction may require less than 1 μM reporter. An electrochemical DETECTR reaction may require less than 100 nM reporter. An electrochemical DETECTR reaction may require less than 10 nM reporter. An electrochemical DETECTR reaction may require less than 1 nM reporter. An electrochemical DETECTR reaction may require less than 100 pM reporter. An electrochemical DETECTR reaction may require less than 10 pM reporter. An electrochemical DETECTR reaction may require less than 1 pM reporter.
Other types of signal amplification that use enrichment may also be used apart from biotin-streptavidin excitation. Non-limiting examples are: (1) glutathione, glutathione S-transferase, (2) maltose, maltose-binding protein, (3) chitin, chitin-binding protein.
Decrease in signal. The progress of the programmable nuclease cleavage reaction may be monitored by recording the decrease in the current produced by a ferrocene (Fc), or other electroactive mediator moieties, conjugated to the individual nucleotides of nucleic acid molecules (ssRNA, ssDNA or ssRNA/DNA hybrid molecules) immobilized on the bottom surface of the detection and incubation chamber. In the absence of the amplified target, the programmable nuclease complex remains inactive, and a high current caused by the electroactive moieties is recorded. When the programmable nuclease complex with guides flows in the detection and incubation chamber and is activated by the matching nucleic acid target at 37° C., the programmable nuclease complex non-specifically degrades the immobilized Fc-conjugated nucleic acid molecules. This cleavage reaction decreases the number of electroactive molecules and, thus, leads to a decrease in recorded current.
The electrochemical detection may also be multiplexed. This is achieved by the addition of one or more working electrodes in the incubation and detection chamber (C1 or C2). The electrodes can be plain, or modified, as described above for the single electrochemical detection method.
Electrochemiluminescence in a combined optical and electrochemical readout method. The optical signal may be produced by luminescence of a compound, such as tri-propyl amine (TPA) generated as an oxidation product of an electroactive product, such as ruthenium bipyridine,[Ru (py)3]2+.
A number of different programmable nuclease proteins may be multiplexed by: (1) separate fluidic paths (parallelization of channels), mixed with the same sample, for each of the proteins, or (2) switching to digital (two-phase) microfluidics, where each individual droplet contains a separate reaction mix. The droplets could be generated from single or double emulsions of water and oil. The emulsions are compatible with programmable nuclease reaction, and optically inert.
The following methods may be used to couple the readout of the Cas reaction to invertase activity:
Colorimetry using a camera, standalone, or an integrated mobile phone optical sensor. The amount of fructose and glucose is linked to a colorimetric reaction. Two examples are: (a) 3,5-Dinitrosalicylic acid (DNS), and (b) formazan dye thiazolyl blue. The color change can be monitored using a CCD camera, or the image sensor of a mobile phone. For this method, we use a variation of the fluidic device described in
Amperometry using a conventional glucometer, or an electrochemical analyzer. A variation of the fluidic device described in
A number of different devices are compatible with detection of target nucleic acids using the methods and compositions disclosed herein. In some embodiments, the device is any of the microfluidic devices disclosed herein. In other embodiments, the device is a lateral flow test strip connected to a reaction chamber. In further embodiments, the lateral flow strip may be connected to a sample preparation device.
In some embodiments, the fluidic device may be a pneumatic device. The pneumatic device may comprise one or more sample chambers connected to one or more detection chambers by one or more pneumatic valves. Optionally, the pneumatic device may further comprise one or more amplification chamber between the one or more sample chambers and the one or more detection chambers. The one or more amplification chambers may be connected to the one or more sample chambers and the one or more detection chambers by one or more pneumatic valves. A pneumatic valve may be made from PDMS, or any other suitable material. A pneumatic valve may comprise a channel perpendicular to a microfluidic channel connecting the chambers and allowing fluid to pass between chambers when the valve is open. In some embodiments, the channel deflects downward upon application of air pressure through the channel perpendicular to the microfluidic channel.
In some embodiments, the fluidic device may be a sliding valve device. The sliding valve device may comprise a sliding layer with one or more channels and a fixed layer with one or more sample chambers and one or more detection chambers. Optionally, the fixed layer may further comprise one or more amplification chambers. In some embodiments, the sliding layer is the upper layer and the fixed layer is the lower layer. In other embodiments, the sliding layer is the lower layer and the fixed layer is the upper layer. In some embodiments, the upper layer is made of a plastic polymer comprising poly-methacrylate (PMMA), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene (PE), high-density polyethylene (HDPE), polypropylene (PP); a glass; or a silicon. In some embodiments, the lower layer is made of a plastic polymer comprising poly-methacrylate (PMMA), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene (PE), high-density polyethylene (HDPE), polypropylene (PP); a glass; or a silicon.The sliding valve device may further comprise one or more of a side channel with an opening aligned with an opening in the sample chamber, a side channel with an opening aligned with an opening in the amplification chamber, or a side channel with an opening aligned with the opening in the detection chamber. In some embodiments the side channels are connected to a mixing chamber to allow transfer of fluid between the chambers. In some embodiments, the sliding valve device comprises a pneumatic pump for mixing, aspirating, and dispensing fluid in the device.
Pneumatic Valve Device. A microfluidic device particularly well suited for carrying out the DETECTR reactions described herein is one comprising a pneumatic valve, also referred to as a “quake valve”. The pneumatic valve can be closed and opened by the flow of air from, for an example, an air manifold. The opening of the pneumatic valve can lead to a downward deflection of the channel comprising the pneumatic valve, which can subsequently deflect downwards and seal off a microfluidic channel beneath the channel comprising the pneumatic valve. This can lead to stoppage of fluid flow in the microfluidic channel. When the air manifold is turned off, the flow of air through the channel comprising the quake valve ceases and the microfluidic channel beneath the channel comprising the quake valve is “open”, and fluid can flow through. In some embodiments, the channel comprising the pneumatic valve may be above or below the microfluidic channel carrying the fluid of interest. In some embodiments, the channel comprising the pneumatic valve can be parallel or perpendicular to the microfluidic channel carrying the fluid of interest. Pneumatic valves can be made of a two hard thermoplastic layers sandwiching a soft silicone layer.
One example layout that is compatible with the compositions and methods disclosed herein is shown in
Sliding Valve Device. A microfluidic device particularly well suited for carrying out the DETECTR reactions described herein is a sliding valve device. The sliding valve device can have a sliding layer and a fixed layer. The sliding layer may be on top and the fixed layer may be on bottom. Alternatively, the sliding layer may be on bottom and the fixed layer may be on top. In some embodiments, the sliding valve has a channel. The channel can have an opening at one end that interacts with an opening in a chamber and the channel can also have an opening at the other end that interacts with an opening in a side channel. In some embodiments, the sliding layer has more than one opening. In some embodiments, the fixed layer comprises a sample chamber, an amplification chamber, and a detection chamber. The sample chamber, the amplification chamber, and the detection layer can all have an opening at the bottom of the chambers. For example, the sample chamber may have an opening for insertion of a sample. When the opening in a chamber is aligned with the opening in a channel, fluid can flow from the chamber into the channel. Further, when the opening in the channel is subsequently aligned with an opening in a side channel, fluid can flow from the channel into the side channel. The side channel can be further fluidically connected to a mixing chamber, or a port in which an instrument (e.g., a pipette pump) for mixing fluid is inserted. Alignment of openings can be enabled by physically moving or automatically actuating the sliding layer to slide along the length of the fixed layer. In some embodiment, the above described pneumatic valves can be added at any position to the sliding valve device in order to control the flow of fluid from one chamber into the next. The sliding valve device can also have multiple layers. For example, the sliding valve can have 2, 3, 4, 5, 6, 7, 8, 9, 10, or more layers.
Lateral Flow Devices. In some embodiments, a device of the present disclosure comprises a chamber and a lateral flow strip.
In some embodiments, the above lateral flow strip can be additionally interfaced with a sample preparation device, as shown in
Resistance Channel Devices. In some embodiments, a device of the present disclosure may resistance channels, sample metering channels, valves for fluid flow or any combination thereof.
A chamber of the device (e.g., the amplification chamber, the detection chamber, or the detection reagent reservoir) may be fluidically connected to one or more additional chambers by one or more channels. In some embodiments, a channel may be a resistance channel configured to regulate the flow of fluid between a first chamber and a second chamber. A resistance channel may form a non-linear path between the first chamber and the second chamber. It may include features to restrict or confound flow, such as bends, turns, fins, chevrons, herringbones or other microstructures. A resistance channel may have reduced backflow compared to a linear channel of comparable length and width. A resistance channel may function by requiring an increased pressure to pass fluid through the channel compared to a linear channel of comparable length and width. In some embodiments, a resistance channel may result in decreased cross-contamination between two chambers connected by the resistance channel as compared to the cross-contamination between two chambers connected by a linear channel of comparable length and width. A resistance channel may have an angular path, for example as illustrated
In some embodiments, a channel may be a sample metering channel. A sample metering channel may form a path between a first chamber and a second chamber and have a channel volume configured to hold a set volume of a fluid to meter the volume of fluid transferred from the first chamber to the second chamber. A sample metering path may form a path between a first chamber and a second chamber and have a channel volume configured to allow to flow from the first channel to the second channel at a desired rate. Metering can also be affected by positive or negative pressure applied to an auxiliary chamber acting as a liquid reagent storage reservoir. This can also be done by storing air in a blister pack for low-cost applications. Examples of sample metering channels are shown in
A schematic example of a resistance channel is shown in
A microfluidic device may comprise one or more reagent ports configured to receive a reagent into the device (e.g., into a chamber of the device). A reagent port may comprise an opening in the wall of a chamber. A reagent port may comprise an opening in the wall of a channel or the end of a channel. A reagent port configured to receive a sample may be a sample inlet port. A reagent (e.g., a buffer, a solution, or a sample) may be introduced into the microfluidic device through a reagent port. The reagent may be introduced manually by a user (e.g., a human user), or the reagent may be introduced automatically by a machine (e.g., by a detection manifold).
A variety of chamber shapes may be utilized in the cartridges of the present disclosure. A chamber may be circular, for example the amplification chambers, detection chambers, and detection reagent reservoirs shown in
A valve may be configured to prevent, regulate, or allow fluid flow from a first chamber to one or more additional chambers. In some embodiments, a valve may rotate from a first position to a second position to prevent, allow, or alter a fluid flow path. In some embodiments, a valve may slide from a first position to a second position to prevent, allow, or alter a fluid flow path. In some embodiments, a valve may open or close based on pressure applied to the valve. In some embodiments, a valve may be an elastomeric valve. The valve can be active (mechanical, non-mechanical, or externally actuated) or passive (mechanical or non-mechanical). A valve may be a push-pull/solenoid actuated valve. A valve may be controlled electronically. For example, a valve may be controlled using a solenoid. In some embodiments, a valve may be controlled manually. Other mechanisms of control may be: magnetic, electric, piezoelectric, thermal, bistable, electrochemical, phase change, rheological, pneumatic, check valving or capillarity. In some embodiment, a valve may be disposable. For example, a valve may be removed from a microfluidic device and replaced with a new valve to prevent contamination when reusing a microfluidic device. In some embodiments, a valve may be covered by a valve cap or elastomeric plug.
The cartridge may be configured to connect to a first pump to pump fluid from the amplification chamber to the detection chamber and to a second pump to pump fluid from the detection reagent reservoir to the detection chamber. A variety of pumps known in the art are functional to move fluid from a first chamber to a second chamber and may be used with a cartridge of the present disclosure. In some embodiments, a cartridge may be used with a peristaltic pump, a pneumatic pump, a hydraulic pump, or a syringe pump.
An example of a microfluidic cartridge is shown in
In some embodiments, a device may comprise a multi-layered, laminated cartridge patterned with laser embossing, and hardware with integrated electronics, optics and mechanics, as shown in
Detection Manifolds. A detection manifold may be used to perform and detect a DETECTR assay of the present disclosure in a device of the present disclosure. A detection manifold may also be referred to herein as a cartridge manifold or a heating manifold. A detection manifold may be configured to facilitate or detect a DETECTR reaction performed in a microfluidic device of the present disclosure. In some embodiments, a detection manifold may comprise one or more heating zones to heat one or more regions of a microfluidic device. In some embodiments, a detection manifold may comprise a first heating zone to heat a first region of a microfluidic device in which an amplification reaction is performed. For example, the first heater may heat the first region of the microfluidic device to about 60° C. In some embodiments, a detection manifold may comprise a second heating zone to heat a second region of a microfluidic device in which a detection reaction is performed. For example, the second heater may heat the second region of the microfluidic device to about 37° C. In some embodiments, a detection manifold may comprise a third heating zone to heat a third region of a microfluidic device in which a lysis reaction is performed. For example, the third heater may heat the third region of the microfluidic device to about 95° C. An example of a detection manifold comprising two insulated heating zones for use with a microfluidic cartridge is shown in
In some embodiments, a detection manifold may comprise an illumination source configured to illuminate a detection chamber of a microfluidic device. The illumination source may be configured to emit a narrow spectrum illumination (e.g., an LED) or the illumination may be configured to emit a broad-spectrum illumination (e.g., an arc lamp). The detection manifold may further comprise one or more filters or gratings to filter for a desired illumination wavelength. In some embodiments, the illumination source may be configured to illuminate a detection chamber (e.g., a chamber comprising a DETECTR reaction) through a top surface of a microfluidic device. In some embodiments, the illumination source may be configured to illuminate a detection chamber through a side surface of a microfluidic device. In some embodiments, the illumination source may be configured to illuminate a detection chamber through a bottom surface of a microfluidic device. In some embodiments, the detection manifold may comprise a sensor for detecting a signal produced by a DETECTR reaction. The signal may be a fluorescent signal. For example, the detection manifold may comprise a camera (e.g., charge-coupled device (CCD), complementary metal-oxide-semiconductor (CMOS)) or a photodiode. A schematic example of a detection manifold is shown in
A detection manifold may comprise electronics configured to control one or more of a temperature, a pump, a valve, an illumination source, or a sensor. In some embodiments, the electronics may be controlled autonomously using a program. For example, the electronics may be autonomously controlled to implement a workflow of the present disclosure (e.g., the workflow provided in
General Features of Devices. In some embodiments, a device of the present disclosure can hold 2 or more amplification chambers. In some embodiments, a device of the present disclosure can hold 10 or more detection chambers. In some embodiments, a device of the present disclosure comprises a single chamber in which sample lysis, target nucleic acid amplification, reverse transcription, and detection are all carried out. In some cases, different buffers are present in the different chambers. In some embodiments, all the chambers of a device of the present disclosure have the same buffer. In some embodiments, the sample chamber comprises the lysis buffer and all of the materials in the amplification and detection chambers are lyophilized or vitrified. In some embodiments, the sample chamber includes any buffer for lysing a sample disclosed herein. The amplification chamber can include any buffer disclosed herein compatible with amplification and/or reverse transcription of target nucleic acids. The detection chamber can include any DETECTR or CRISPR buffer (e.g., an MBuffer) disclosed herein or otherwise capable of allowing DETECTR reactions to be carried out. In this case, once sample lysing has occurred, volume is moved from the sample chamber to the other chambers in an amount enough to rehydrate the materials in the other chambers. In some embodiments, the device further comprises a pipette pump at one end for aspirating, mixing, and dispensing liquids. In some embodiments, an automated instrument is used to control aspirating, mixing, and dispensing liquids. In some embodiments, no other instrument is needed for the fluids in the device to move from chamber to chamber or for sample mixing to occur. A device of the present disclosure may be made of any suitable thermoplastic, such as COC, polymer COP, teflon, or another thermoplastic material. Alternatively, the device may be made of glass. In some embodiments, the detection chamber may include beads, such as nanoparticles (e.g., a gold nanoparticle). In some embodiments, the reporters are immobilized on the beads. In some embodiments, after cleavage from the bead, the liberated reporters flow into a secondary detection chamber, where detection of a generated signal occurs by any one of the instruments disclosed herein. In some embodiments, the detection chamber is shallow, but has a large surface area that is optimized for optical detection. A device of the present disclosure may also be coupled to a thermoregulator. For example, the device may be on top of or adjacent to a planar heater that can heat the device up to high temperatures. Alternatively, a metal rod conducting heat is inserted inside the device and presses upon a soft polymer. The heat is transferred to the sample by dissipating through the polymer and into the sample. This allows for sample heating with direct contact between the metal rod and the sample. In some embodiments, in addition to or in place of a buffer for lysing a sample, the sample chamber may include an ultrasonicator for sample lysis. A swab carrying the sample may be inserted directly into the sample chamber. Commonly, a buccal swab may be used, which can carry blood, urine, or a saliva sample. A filter may be included in any of the chambers in the devices disclosed herein to filter the sample prior to carrying it to the next step of the method. Any of the devices disclosed herein can be couple to an additional sample preparation module for further manipulation of the sample before the various steps of the DETECTR reaction. In some embodiments the reporter can be in solution in the detection chamber. In other embodiments, the reporter can be immobilized directly on the surface of the detection chamber. The surface can be the top or the bottom of the chamber. In still other embodiments, the reporter can be immobilized to the surface of a bead. In the case of a bead, after cleavage, the detectable signal may be washed into a subsequent chamber while the bead remains trapped—thus allowing for separation of the detectable signal from the bead. Alternatively, cleavage of the reporter off of the surface of the bead is enough to generate a strong enough detectable signal to be measured. By sequestering or immobilizing the above described reporters, the stability of the reporters in the devices disclosed herein carrying out DETECTR reactions may be improved. Any of the above devices can be compatible for colorimetric, fluorescence, amperometric, potentiometric, or another electrochemical signal. In some embodiments, the colorimetric, fluorescence, amperometric, potentiometric, or another electrochemical sign may be detected using a measurement device connected to the detection chamber (e.g., a fluorescence measurement device, a spectrophotometer, or an oscilloscope).
In some embodiments, signals themselves can be amplified, for example via use of an enzyme such as horse radish peroxidase (HRP). In some embodiments, biotin and avidin reactions, which bind at a 4:1 ratio can be used to immobilize multiple enzymes or secondary signal molecules (e.g., 4 enzymes of secondary signal molecules, each on a biotin) to a single protein (e.g., avidin). In some embodiments, an electrochemical signal may be produced by an electrochemical molecule (e.g., biotin, ferrocene, digoxigenin, or invertase). In some embodiments, the above devices could be couple with an additional concentration step. For example, silica membranes may be used to capture nucleic acids off a column and directly apply the Cas reaction mixture on top of said filter. In some embodiments, the sample chamber of any one of the devices disclosed herein can hold from 20 ul to 1000 ul of volume. In some embodiments, the sample chamber holds from 20 to 500, from 40 to 400, from 30 to 300, from 20 to 200 or from 10 to 100 ul of volume. In preferred embodiments, the sample chamber holds 200 ul of volume. The amplification and detection chambers can hold a lower volume than the sample chamber. For example, the amplification and detection chambers may hold from 1 to 50, 10 to 40, 20 to 30, 10 to 40, 5 to 35, 40 to 50, or 1 to 30 ul of volume. Preferably, the amplification and detection chambers may hold about 200 ul of volume. In some embodiments, an exonuclease is present in the amplification chamber or may be added to the amplification chamber. The exonuclease can clean up single stranded nucleic acids that are not the target. In some embodiments, primers for the target nucleic acid can be phosophorothioated in order to prevent degradation of the target nucleic acid in the presence of the exonuclease. In some embodiments, any of the devices disclosed herein can have a pH balancing well for balancing the pH of a sample. In some embodiments, in each of the above devices, the reporter is present in at least four-fold excess of total nucleic acids (target nucleic acids +non-target nucleic acids). Preferably the reporter is present in at least 10-fold excess of total nucleic acids. In some embodiments, the reporter is present in at least 4-fold, at least 5-fold at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, from 1.5 to 100-fold, from 4 to 80-fold, from 4 to 10-fold, from 5 to 20-fold or from 4 to 15-fold excess of total nucleic acids. In some embodiments, any of the devices disclosed herein can carry out a DETECTR reaction with a limit of detection of at least 0.1 aM, at least 0.1 nM, at least 1 nM or from 0.1 aM to 1 nM. In some embodiments, the devices disclosed herein can carry out a DETECTR reaction with a positive predictive value of at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100%. In some embodiments, the devices disclosed herein can carry out a DETECTR reaction with a negative predictive value of at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100%. In some embodiments, spatial multiplexing in the above devices is carried out by having at least one, more than one, or every detection chamber in the device comprise a unique guide nucleic acid.
Workflows. A DETECTR reaction may be performed in a microfluidic device using many different workflows. In some embodiments, a workflow for measuring a buccal swab sample may comprise swabbing a cheek, adding the swab to a lysis solution, incubating the swab to lyse the sample, combining the lysed sample with reagents for amplification of a target nucleic acid, combining the amplified sample with DETCTR reagents, and incubating the sample to detect the target nucleic acid. In some embodiments, one or more of lysis, amplification, and detection may be performed in a microfluidic device (e.g., a microfluidic cartridge illustrated in
An example of a workflow for detecting a target nucleic acid is provided in
An example of a workflow for detecting a target nucleic acid (e.g., a viral target nucleic acid) may comprise swabbing a cheek of a subject. The swab may be added to about 200 μL of a low-pH solution. In some embodiments, the swab may displace the solution so that the total volume is about 220 μL. The swab may be incubated in the low-pH solution for about a minute. In some embodiments, cells or viral capsids present on the swab may be lysed in the low-pH solution. A portion of the sample (5 μL) may be combined with about 45 μL of an amplification solution in an amplification chamber. The total volume within the chamber may be about 50 μL. The sample may be incubated in the amplification chamber for up to about 30 minutes at a temperature of from about 50° C. to about 65° C. to amplify the target nucleic acid the sample. In some embodiments, two aliquots of about 5 μL each of the amplified sample may be directed to two detection chambers where they are combined with about 95 μL each of a DETECTR reaction mix. The amplified sample may be incubated with the DETECTR reaction mix for up to about 10 minutes at about 37° C. in each of two detection chambers to detect the presence or absence of the target nucleic acid.
In some embodiments, a workflow for a DETECTR reaction performed in a microfluidic device may be implemented by a user. A user may collect a sample from a subject (e.g., a buccal swab or a nasal swab), place the sample in a lysis buffer, add the lysed sample to a microfluidic cartridge of the present disclosure, and insert the cartridge in a detection manifold of the present disclosure. In some embodiments, a user may add an unlysed sample to the microfluidic cartridge. In some embodiments, a workflow for a DETECTR reaction may be implemented in a microfluidic cartridge of the present disclosure. A microfluidic cartridge may comprise one or more reagents in one or more chambers to facilitate one or more of lysis, amplification, or detection of a target nucleic acid in a sample. In some embodiments, a workflow for a DETECTR reaction performed in a microfluidic device may be facilitated by a detection manifold. A detection manifold may provide one or more of heating control for an amplification reaction, a detection reaction, or both, solution movement control (e.g., pump control or valve control), illumination, or detection.
In some embodiments, a workflow for a DETECTR performed a microfluidic cartridge and facilitated by a user and a detection manifold may comprise steps of: 1) user loads sample into cartridge comprising one or more reagents, 2) user inserts cartridge into a detection manifold and presses a start button, 3) manifold energizes a solenoid to close a valve between a amplification chamber and a detection chamber, 4) manifold indicator LED turns on, 5) manifold turns on first heater to heat a first heating zone to 60° C. and second heater to heat a second heating zone to 37° C., 5) incubate sample in amplification chamber for 30 minutes in first heating zone to amplify sample, 6) manifold turns off first heater, 7) manifold de-energizes solenoid to open valve, 8) manifold turns on a first pump for 15 seconds to pump the amplified sample to the detection chamber, 9) manifold turns off first pump, 10) manifold turns on a second pump for 15 seconds to pump detection reagents from a detection reagent storage chamber to the detection chamber, 11) manifold turns off second pump, 12) incubate amplified sample and detection reagents in detection chamber for 30 minutes in second heating zone to perform detection reaction, 13) manifold indicator LED turns off, 14) manifold turns on illumination source and measures detectable signal produced by detection reaction.
An example of a workflow that may be performed in a microfluidic device, for example the microfluidic device shown in
In some embodiments, a workflow performed in microfluidic device may comprise partitioning a sample into two or more chambers. A device may be configured to partition a sample into a plurality of portions. A device may be configured to transfer two portions of a partitioned sample into separate fluidic channels or chambers. A device may be configured to transfer a plurality of portions of a sample into a plurality of different fluidic channels or chambers. A device may be configured to perform reactions on individual portions of a partitioned sample. A device may be configured to partition a sample into 2 portions. A device may be configured to partition a sample into 3 portions. A device may be configured to partition a sample into 4 portions. A device may be configured to partition a sample into 5 portions. A device may be configured to partition a sample into 6 portions. A device may be configured to partition a sample into 7 portions. A device may be configured to partition a sample into 8 portions. A device may be configured to partition a sample into 9 portions. A device may be configured to partition a sample into 10 portions. A device may be configured to partition a sample into 12 portions. A device may be configured to partition a sample into 15 portions. A device may be configured to divide a sample into at least 20 portions. A device may be configured to partition a sample into at least 50 portions. A device may be configured to partition a sample into 100 portions. A device may be configured to partition a sample into 500 portions.
A device may be configured to perform a first reaction on a first portion of a sample and a second reaction on a second portion of a partitioned sample. A device may be configured to perform a different reaction on each portion of a partitioned sample. A device may be configured to perform sequential reactions on a sample or a portion of a sample. A device may be configured to perform a first reaction in a first chamber and a second reaction in a second chamber on a sample or portion of a sample.
A device may be configured to mix a sample with reagents. In some cases, a device mixes a sample with reagents by flowing the sample and reagents back and forth between a plurality of compartments. In some cases, a device mixes a sample with reagents by cascading the sample and reagents into a single compartment (e.g., by flowing both the sample and reagents into the compartment from above). In some cases, the mixing method performed by the device minimizes the formation of bubbles. In some cases, the mixing method performed by the device minimizes the sample loss or damage (e.g., protein precipitation).
A device may be configured to perform a plurality of reactions on a plurality of portions of a sample. In some cases, a device comprises a plurality of chambers each comprising reagents. In some cases, two chambers from among the plurality of reagent comprising chambers comprise different reagents. In some cases, a first portion and a second portion of a sample may be subjected to different reactions. In some cases, a first portion and a second portion of a sample may be subjected to the same reactions in the presence of different reporter molecules. In some cases, a first portion and a second portion of a sample may be subjected to the same detection method. In some cases, a first portion and a second portion of a sample may be subjected to different detection methods. In some cases, a plurality of portions of a sample may be detected separately (e.g., by a diode array that excites and detects fluorescence from each portion of a sample individually). In some cases, a plurality of portions of a sample may be detected simultaneously. For example, a device may partition a single sample into 4 portions, perform different amplification reactions on each portion, partition the products of each amplification reaction into two portions, perform different DETECTR reactions on each portion, and individually measure the progress of each DETECTR reaction.
A device may be configured to partition a small quantity of sample for a large number of different reactions or sequences of reactions. In some cases, a device may partition less than 1 ml of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 800 μl of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 600 μl of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 400 μl of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 200 μl of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 100 μl of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 50 μl of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 1 mg of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 800 μg of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 600 μg of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 400 μg of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 200 μg of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 100 μg of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 50 μg of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 20 μg of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 10 μg of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 1 μg of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 800 ng of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 600 ng of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 400 ng of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 200 ng of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 100 ng of sample for a plurality of different reactions or sequences of reactions. In some cases, a device may partition less than 50 ng of sample for a plurality of different reactions or sequences of reactions. In some cases, the sample may comprise nucleic acid. In some cases, the sample may comprise cells. In some cases, the sample may comprise proteins. In some cases, the plurality of different reactions or sequences of reactions may comprise 2 or more different reactions or sequences of reactions. In some cases, the plurality of different reactions or sequences of reactions may comprise 3 or more different reactions or sequences of reactions. In some cases, the plurality of different reactions or sequences of reactions may comprise 4 or more different reactions or sequences of reactions. In some cases, the plurality of different reactions or sequences of reactions may comprise 5 or more different reactions or sequences of reactions. In some cases, the plurality of different reactions or sequences of reactions may comprise 10 or more different reactions or sequences of reactions. In some cases, the plurality of different reactions or sequences of reactions may comprise 20 or more different reactions or sequences of reactions. In some cases, the plurality of different reactions or sequences of reactions may comprise 50 or more different reactions or sequences of reactions. In some cases, the plurality of different reactions or sequences of reactions may comprise 100 or more different reactions or sequences of reactions. In some cases, the plurality of different reactions or sequences of reactions may comprise 500 or more different reactions or sequences of reactions. In some cases, the plurality of different reactions or sequences of reactions may comprise 1000 or more different reactions or sequences of reactions. In some cases, a first reaction or sequence of reactions and a second reaction or sequence of reactions detect two different nucleic acid sequences. In some cases, each reaction or sequence of reactions from among a plurality of different reactions or sequences of reactions detects a different nucleic acid sequence. For example, a device may be configured to perform 40 different sequences of reactions designed to detect 40 different nucleic acid sequences from a single sample comprising 200 ng DNA (e.g., 200 ng DNA from a buccal swab). In such a case, each of the 40 different nucleic acid sequences could be used to determine the presence of a particular virus in the sample.
In some cases, a device is configured to automate a step. In some cases, a device automates a sample partitioning step. In some cases, a device automates a reaction step (e.g., by mixing a sample with reagents and heating to a temperature for a defined length of time). In some cases, the device automates every step following sample input. In some cases, a device may automate a plurality of reactions on a single input sample. In some cases, a device may automate, detect, and provide results for a plurality of reactions on a single input sample. In some cases, a device may automate, detect, and provide results for a plurality of reactions on a single sample in less than 2 hours. For example, a device may automate 100 separate amplification and DETECTR reactions on a sample comprising 400 ng DNA, detect and then provide the results of the reactions in less than 2 hours. In some cases, a device may automate, detect, and provide results for a plurality of reactions on a single sample in less than 1 hour. In some cases, a device may automate, detect, and provide results for a plurality of reactions on a single sample in less than 40 minutes. In some cases, a device may automate, detect, and provide results for a plurality of reactions on a single sample in less than 20 minutes. In some cases, a device may automate, detect, and provide results for a plurality of reactions on a single sample in less than 10 minutes. In some cases, a device may automate, detect, and provide results for a plurality of reactions on a single sample in less than 5 minutes. In some cases, a device may automate, detect, and provide results for a plurality of reactions on a single sample in less than 2 minutes.
Microfluidic devices and detection manifolds for detection of viral infections. A microfluidic device of the present disclosure (e.g., a microfluidic device illustrated in
A microfluidic device of the present disclosure (e.g., a microfluidic device illustrated in
A microfluidic device of the present disclosure (e.g., a microfluidic device illustrated in
Disclosed herein are kits fluidic devices, and systems for use to detect a target nucleic acid. In some embodiments, the kit comprises the reagents and the support medium. The reagent may be provided in a reagent chamber or on the support medium. Alternatively, the reagent may be placed into the reagent chamber or the support medium by the individual using the kit. Optionally, the kit further comprises a buffer and a dropper. The reagent chamber be a test well or container. The opening of the reagent chamber may be large enough to accommodate the support medium. The buffer may be provided in a dropper bottle for ease of dispensing. The dropper can be disposable and transfer a fixed volume. The dropper can be used to place a sample into the reagent chamber or on the support medium.
In some embodiments, a kit for detecting a target nucleic acid comprising a support medium; a guide nucleic acid targeting a target nucleic acid segment; a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment; and a single stranded detector nucleic acid comprising a detection moiety, wherein the detector nucleic acid is capable of being cleaved by the activated nuclease, thereby generating a first detectable signal.
In some embodiments, a kit for detecting a target nucleic acid comprising a PCR plate; a guide nucleic acid targeting a target nucleic acid segment; a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment;
and a single stranded detector nucleic acid comprising a detection moiety, wherein the detector nucleic acid is capable of being cleaved by the activated nuclease, thereby generating a first detectable signal. The wells of the PCR plate can be pre-aliquoted with the guide nucleic acid targeting a target nucleic acid segment, a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target sequence, and at least one population of a single stranded detector nucleic acid comprising a detection moiety. A user can thus add the biological sample of interest to a well of the pre-aliquoted PCR plate and measure for the detectable signal with a fluorescent light reader or a visible light reader.
In some instances, such kits may include a package, carrier, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, test wells, bottles, vials, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass, plastic, or polymers.
The kit or systems described herein contain packaging materials. Examples of packaging materials include, but are not limited to, pouches, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for intended mode of use.
A kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included. In one embodiment, a label is on or associated with the container. In some instances, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.
After packaging the formed product and wrapping or boxing to maintain a sterile barrier, the product may be terminally sterilized by heat sterilization, gas sterilization, gamma irradiation, or by electron beam sterilization. Alternatively, the product may be prepared and packaged by aseptic processing.
Disclosed herein are stable compositions of the reagents and the programmable nuclease system for use in the methods as discussed above. The reagents and programmable nuclease system described herein may be stable in various storage conditions including refrigerated, ambient, and accelerated conditions. Disclosed herein are stable reagents. The stability may be measured for the reagents and programmable nuclease system themselves or the reagents and programmable nuclease system present on the support medium.
In some instances, stable as used herein refers to a reagents having about 5% w/w or less total impurities at the end of a given storage period. Stability may be assessed by HPLC or any other known testing method. The stable reagents may have about 10% w/w, about 5% w/w, about 4% w/w, about 3% w/w, about 2% w/w, about 1% w/w, or about 0.5% w/w total impurities at the end of a given storage period.
In some embodiments, stable as used herein refers to a reagents and programmable nuclease system having about 10% or less loss of detection activity at the end of a given storage period and at a given storage condition. Detection activity can be assessed by known positive sample using a known method. Alternatively or combination, detection activity can be assessed by the sensitivity, accuracy, or specificity. In some embodiments, the stable reagents has about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.5% loss of detection activity at the end of a given storage period.
In some embodiments, the stable composition has zero loss of detection activity at the end of a given storage period and at a given storage condition. The given storage condition may comprise humidity of equal to or less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative humidity. The controlled storage environment may comprise humidity between 0% and 50% relative humidity, 0% and 40% relative humidity, 0% and 30% relative humidity, 0% and 20% relative humidity, or 0% and 10% relative humidity. The controlled storage environment may comprise temperatures of −100° C., −80° C., −20° C., 4° C., about 25° C. (room temperature), or 40° C. The controlled storage environment may comprise temperatures between −80° C. and 25° C., or −100° C. and 40° C. The controlled storage environment may protect the system or kit from light or from mechanical damage. The controlled storage environment may be sterile or aseptic or maintain the sterility of the light conduit. The controlled storage environment may be aseptic or sterile.
In some cases, reagents may be stored in a capillary. A capillary may be a glass capillary. In some cases, a capillary provides a controlled storage environment. A capillary may also be stored within a controlled storage environment. A capillary can store a solution containing a reagent. A capillary can store a reagent in a dry form. A capillary can be loaded with a solution containing a reagent and then be dried to yield a capillary containing a dried or powdered form of the reagent. A dried or powdered reagent may be hydrated or dissolved by filling the capillary with a solution (e.g., buffer). A reagent within a capillary may be stable when stored at room temperature. A reagent within a capillary may stable when stored at (e.g., 37° C.). A reagent within a capillary may be stable when stored below room temperature (e.g., 4 37° C.). A reagent within a capillary may be stable when stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. A reagent stored within a capillary may be stable when stored for longer than a year. A reagent stored within a capillary may retain greater than 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of its activity.
A capillary can contain an enzyme in dried form or in solution. A capillary can contain a programmable nuclease in dried form or in solution. A capillary can contain a nucleic acid in dried form or in solution. A capillary can contain an ribonucleoprotein in dried form or in solution. A capillary can contain a dye in dried form or in solution. A capillary can contain a buffer (e.g., a lysis buffer) in dried form or in solution. A capillary can contain amplification reagents in dried form or in solution.
A reagent may be removed from a capillary by flowing a solution through the capillary. A reagent may be removed from a capillary by applying pressure (e.g., hydraulic or pneumatic pressure) to an open end of the capillary. A reagent may be removed from a capillary by breaking the capillary. A capillary may be positioned so that its contents elute due to gravity. A capillary may be open at both ends. A capillary may be sealed at one or two ends.
A capillary may have an internal volume of less than 1 μl. A capillary can have an internal volume of 1 μl. A capillary can have an internal volume of 2 μl. A capillary can have an internal volume of 3 μl. A capillary can have an internal volume of 4 μl. A capillary can have an internal volume of 5 μl. A capillary can have an internal volume of between 5 and 10 μl. A capillary can have an internal volume of between 10 and 20 μl. A capillary can have an internal volume of between 20 and 30 μl. A capillary can have an internal volume of between 30 and 40 μl. A capillary can have an internal volume of between 40 and 50 μl. A capillary can have an internal volume of between 50 and 60 μl. A capillary can have an internal volume of between 60 and 70 μl. A capillary can have an internal volume of between 70 and 80 μl. A capillary can have an internal volume of between 80 and 90 μl. A capillary can have an internal volume of between 90 and 100 μl. A capillary can have an internal volume of greater than 100 μl.
The kit or system can be packaged to be stored for extended periods of time prior to use. The kit or system may be packaged to avoid degradation of the kit or system. The packaging may include desiccants or other agents to control the humidity within the packaging. The packaging may protect the kit or system from mechanical damage or thermal damage. The packaging may protect the kit or system from contamination of the reagents and programmable nuclease system. The kit or system may be transported under conditions similar to the storage conditions that result in high stability of the reagent or little loss of reagent activity. The packaging may be configured to provide and maintain sterility of the kit or system. The kit or system can be compatible with standard manufacturing and shipping operations.
A number of target amplification and detection methods are consistent with the methods, compositions, reagents, enzymes, and kits disclosed herein. As described herein, a target nucleic acid may be detected using a DNA-activated programmable RNA nuclease (e.g., a Cas13), a DNA-activated programmable DNA nuclease (e.g., a Cas12), or an RNA-activated programmable RNA nuclease (e.g., a Cas13) and other reagents disclosed herein (e.g., RNA components). The target nucleic acid may be detected using DETECTR, as described herein. The target nucleic acid may be an RNA, reverse transcribed RNA, DNA, DNA amplicon, amplified DNA, synthetic nucleic acids, or nucleic acids found in biological or environmental samples. In some cases, the target nucleic acid is amplified prior to or concurrent with detection. In some cases, the target nucleic acid is reverse transcribed prior to amplification. The target nucleic acid may be amplified via loop mediated isothermal amplification (LAMP) of a target nucleic acid sequence. In some cases, the nucleic acid is amplified using LAMP coupled with reverse transcription (RT-LAMP). The LAMP amplification may be performed independently, or the LAMP amplification may be coupled to DETECTR for detection of the target nucleic acid. The RT-LAMP amplification may be performed independently, or the RT-LAMP amplification may be coupled to DETECTR for detection of the target nucleic acid. The DETECTR reaction may be performed using any method consistent with the methods disclosed herein.
In some embodiments, a LAMP amplification reaction comprises a plurality of primers, dNTPs, and a DNA polymerase. LAMP may be used to amplify DNA with high specificity under isothermal conditions. The DNA may be single stranded DNA or double stranded DNA. In some cases, a target nucleic acid comprising RNA may be reverse transcribed into DNA using a reverse transcriptase prior to LAMP amplification. A reverse transcription reaction may comprise primers, dNTPs, and a reverse transcriptase. In some cases, the reverse transcription reaction and the LAMP amplification reaction may be performed in the same reaction. A combined RT-LAMP reaction may comprise LAMP primers, reverse transcription primers, dNTPs, a reverse transcriptase, and a DNA polymerase. In some case, the LAMP primers may comprise the reverse transcription primers.
A DETECTR reaction to detect the target nucleic acid sequence may comprise a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease. The programmable nuclease when activated, as described elsewhere herein, exhibits sequence-independent cleavage of a reporter (e.g., a nucleic acid comprising a moiety that becomes detectable upon cleavage of the nucleic acid by the programmable nuclease). The programmable nuclease is activated upon the guide nucleic acid hybridizing to the the target nucleic acid. A combined LAMP DETECTR reaction may comprise a plurality of primers, dNTPs, a DNA polymerase, a guide nucleic acid, a programmable nuclease, and a substrate nucleic acid. A combined RT-LAMP DETECTR reaction may comprise LAMP primers, reverse transcription primers, dNTPs, a reverse transcriptase, a DNA polymerase, a guide nucleic acid, a programmable nuclease, and a substrate nucleic acid. In some case, the LAMP primers may comprise the reverse transcription primers. LAMP and DETECTR can be carried out in the same sample volume. LAMP and DETECTR can be carried out concurrently in separate sample volumes or in the same sample volume. RT-LAMP and DETECTR can be carried out in the same sample volume. RT-LAMP and DETECTR can be carried out concurrently in separate sample volumes or in the same sample volume.
A LAMP reaction may comprise a plurality of primers. A plurality of primers are designed to amplify a target nucleic acid sequence, which is shown in
Further, as shown in
A set of LAMP primers may be designed for use in combination with a DETECTR reaction. The nucleic acid may comprise a region (e.g., a target nucleic acid), to which a guide RNA hybridizes. All or part of the guide RNA sequence may be reverse complementary to all or part of the target sequence. The target nucleic acid sequence may be adjacent to a protospacer adjacent motif (PAM) 3′ of the target nucleic acid sequence. The PAM may promote interaction the programmable nuclease with the target nucleic acid. The target nucleic acid sequence may be adjacent to a protospacer flanking site (PFS) 3′ of the target nucleic acid sequence. The PFS may promote interaction the programmable nuclease with the target nucleic acid. One or more of the guide RNA, the PAM or PFS, or the target nucleic acid sequence may be specifically positioned with respect to one or more of the F1, F1c, F2, F2c, F3, F3c, LF, LFc, LB, LBc, B1, B1c, B2, B2c, B3, and/or B3c regions.
In some cases, the guide RNA is reverse complementary to a sequence of the target nucleic acid, which is between an F1c region and a B1 region, as in
In some cases, the guide RNA is partially reverse complementary to a sequence of the target nucleic acid, which is between an F1c region and a B1 region, as in
In some cases, the guide RNA is reverse complementary to no more than 50%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, or no more than 5% of the forward inner primer, the backward inner primer, or a combination thereof the sequence between the F1c region and the B1 region or the sequence between the B1c region and the F1 region is at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 99%, or 100% reverse complementary to the guide nucleic acid sequence. In some cases, the guide nucleic acid has a sequence reverse complementary to no more than 50%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, or no more than 5% of the forward inner primer, the backward inner primer, the forward outer primer, the backward outer primer, or any combination thereof. In some cases, the guide nucleic acid sequence has a sequence reverse complementary to no more than 50%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, or no more than 5% of a sequence of an F3c region, an F2c region, the F1c region, the B1c region, an B2c region, an B3c region, or any combination thereof.
In some cases, the region corresponding to the guide RNA sequence does not overlap or hybridize to any of the primers and may further not overlap with or hybridize to any of the regions shown in
In some cases, all or a portion of the guide nucleic acid is reverse complementary to a sequence of the target nucleic acid in a loop region. For example, all or a portion of the sequence of the target nucleic acid that hybridizes to the gRNA may be located between the B1c and B2 regions, as shown in
In some cases, a LAMP primer set may be designed using a commercially available primer design software. A LAMP primer set may be designed for use in combination with a DETECR reaction, a reverse transcription reaction, or both. In some cases, a LAMP primer set may be designed using distributed ledger technology (DLT), artificial intelligence (AI), extended reality (XR) and quantum computing, commonly called “DARQ.” In some cases, a LAMP primer set may be designed using quenching of unincorporated amplification signal reporters (QUASR) (Ball et al., Anal Chem. 2016 Apr. 5; 88(7):3562-8. doi: 10.1021/acs.analchem.5b04054. Epub 2016 Mar. 24.). These methods of designing a set of LAMP primers are provided by way of example only; other methods of designing a set of LAMP primers may be readily apparent to one skilled in the art and may be employed in any of the compositions, kits and methods described herein. Exemplary sets of LAMP primers for use in a combined RT-LAMP DETECTR reaction or LAMP-DETECTR to detect the presence of a nucleic acid sequence corresponding to a respiratory syncytial virus (RSV), an influenza A virus (IAV), an influenza B virus (IAV), or a HERC2 SNP are provided in TABLE 6.
A set of LAMP primers may be designed for use in combination with a DETECTR reaction to detect a single nucleotide polymorphism (SNP) in a target nucleic acid. In some embodiments, a sequence of the target nucleic acid comprising the SNP may be reverse complementary to all or a portion of the guide nucleic acid. For example, the SNP may be positioned within a sequence of the target nucleic acid that is reverse complementary to the guide RNA sequence, as illustrated in
A DETECTR reaction may be used to detect the presence of a specific single nucleotide polymorphism (SNP) allele in a sample. The DETECTR reaction may produce a detectable signal, as described elsewhere herein, in the presence of a target nucleic acid comprising a specific SNP allele. The DETECTR reaction may not produce a signal in the absence of the target nucleic acid or in the presence of a nucleic acid sequence that does not comprise the specific SNP allele or comprises a different SNP allele. In some cases, a DETECTR reaction may comprise a guide RNA reverse complementary to a portion of a target nucleic acid sequence comprising a specific SNP allele. The guide RNA and the target nucleic acid comprising the specific SNP allele may bind to and activate a programmable nuclease, thereby producing a detectable signal as described elsewhere herein. The guide RNA and a nucleic acid sequence that does not comprise the specific SNP allele may not bind to or activate the programmable nuclease and may not produce a detectable signal. In some cases, a target nucleic acid sequence that may or may not comprise a specific SNP allele may be amplified using, for example, a LAMP amplification reaction. In some cases, the LAMP amplification reaction may be combined with a reverse transcription reaction, a DETECTR reaction, or both. For example, the LAMP reaction may be an RT-LAMP reaction, a LAMP DETECTR reaction, or an RT-LAMP DETECTR reactions.
A DETECTR reaction, as described elsewhere herein, may produce a detectable signal specifically in the presence of a target nucleic acid sequence comprising a specific SNP allele. For example, the DETECTR reaction may produce a detectable signal in the presence of a target nucleic acid comprising a G nucleic acid at a location of a SNP but not in the presence of a nucleic acid comprising a C, a T, or an A nucleic acid at the location of the SNP. The DETECTR reaction may produce a detectable signal in the presence of a target nucleic acid comprising a T nucleic acid at a location of a SNP but not in the presence of a nucleic acid comprising a G, a C, or an A nucleic acid at the location of the SNP. The DETECTR reaction may produce a detectable signal in the presence of a target nucleic acid comprising a C nucleic acid at a location of a SNP but not in the presence of a nucleic acid comprising a G, a T, or an A nucleic acid at the location of the SNP. The DETECTR reaction may produce a detectable signal in the presence of a target nucleic acid comprising an A nucleic acid at a location of a SNP but not in the presence of a nucleic acid comprising a G, a T, or a C nucleic acid at the location of the SNP. In addition to the DETECTR reaction, the target nucleic acid having the SNP may be concurrently, sequentially, concurrently together in a sample, or sequentially together in a sample be carried out alongside LAMP or RT-LAMP. For example, the reactions can comprise LAMP and DETECTR reactions, or RT-LAMP and DETECTR reactions. Performing a DETECTR reaction in combination with a LAMP reaction may result in an increased detectable signal as compared to the DETECTR reaction in the absence of the LAMP reaction.
In some cases, the detectable signal produced in the DETECTR reaction may be higher in the presence of a target nucleic acid comprising a specific SNP allele than in the presence of a nucleic acid that does not comprise the specific SNP allele. In some cases, the DETECTR reaction may produce a detectable signal that is at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at last 400-fold, at least 500-fold, at least 1000-fold, at least 2000-fold, at least 3000-fold, at least 4000-fold, at least 5000-fold, at least 6000-fold, at least 7000-fold, at least 8000-fold, at least 9000-fold, at least 10000-fold, at least 50000-fold, at least 100000-fold, at least 500000-fold, or at least 1000000-fold greater in the presence of a target nucleic acid comprising a specific SNP allele than in the presence of a nucleic acid that does not comprise the specific SNP allele. In some cases, the DETECTR reaction may produce a detectable signal that is from 1-fold to 2-fold, from 2-fold to 3-fold, from 3-fold to 4-fold, from 4-fold to 5-fold, from 5-fold to 10-fold, from 10-fold to 20-fold, from 20-fold to 30-fold, from 30-fold to 40-fold, from 40-fold to 50-fold, from 50-fold to 100-fold, from 100-fold to 500-fold, from 500-fold to 1000-fold, from 1000-fold to 10,000-fold, from 10,000-fold to 100,000-fold, or from 100,000-fold to 1,000,000-fold greater in the presence of a target nucleic acid comprising a specific SNP allele than in the presence of a nucleic acid that does not comprise the specific SNP allele.
A DETECTR reaction may be used to detect the presence of a SNP allele associated with a disease or a condition in a nucleic acid sample. The DETECTR reaction may be used to detect the presence of a SNP allele associated with an increased likelihood of developing a disease or a condition in a nucleic acid sample. The DETECTR reaction may be used to detect the presence of a SNP allele associated with a phenotype in a nucleic acid sample. For example, a DETECTR reaction may be used to detect a SNP allele associated with a disease such as phenylketonuria (PKU), cystic fibrosis, sickle-cell anemia, albinism, Huntington's disease, myotonic dystrophy type 1, hypercholesterolemia, neurofibromatosis, polycystic kidney disease, hemophilia, muscular dystrophy, hypophosphatemic rickets, Rat's syndrome, or spermatogenic failure. A DETECTR reaction may be used to detect a SNP allele associated with an increased risk of cancer, for example bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, gallbladder cancer, stomach cancer, leukemia, liver cancer, lung cancer, oral cancer, esophageal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, testicular cancer, thyroid cancer, neuroblastoma, or lymphoma. A DETECTR reaction may be used to detect a SNP allele associated with an increased risk of a disease, for example Alzheimer's disease, Parkinson's disease, amyloidosis, heterochromatosis, celiac disease, macular degeneration, or hypercholesterolemia. A DETECTR reaction may be used to detect a SNP allele associated with a phenotype, for example, eye color, hair color, height, skin color, race, alcohol flush reaction, caffeine consumption, deep sleep, genetic weight, lactose intolerance, muscle composition, saturated fat and weight, or sleep movement.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated.
As used herein, the term “comprising” and its grammatical equivalents specifies the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.
Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers +/−10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.
As used herein the terms “individual,” “subject,” and “patient” are used interchangeably and include any member of the animal kingdom, including humans.
As used herein the term “antibody” refers to, but not limited to, a monoclonal antibody, a synthetic antibody, a polyclonal antibody, a multispecific antibody (including a bi-specific antibody), a human antibody, a humanized antibody, a chimeric antibody, a single-chain Fvs (scFv) (including bi-specific scFvs), a single chain antibody, a Fab fragment, a F(ab′) fragment, a disulfide-linked Fvs (sdFv), or an epitope-binding fragment thereof. In some cases, the antibody is an immunoglobulin molecule or an immunologically active portion of an immunoglobulin molecule. In some instances, an antibody is animal in origin including birds and mammals. Alternately, an antibody is human or a humanized monoclonal antibody.
While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
The following embodiments recite non-limiting permutations of combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is contemplated as depending from or relating to every previous or subsequent numbered embodiment, independent of their order as listed. 1. A microfluidic cartridge for detecting a target nucleic acid comprising: a) an amplification chamber fluidically connected to a valve; b) a detection chamber fluidically connected to the valve, wherein the valve is connected to a sample metering channel; c) a detection reagent chamber fluidically connected to the detection chamber via a resistance channel, the detection reagent chamber comprising a programmable nuclease, a guide nucleic acid, and a labeled detector nucleic acid, wherein the labeled detector nucleic acid is capable of being cleaved upon binding of the guide nucleic acid to a segment of a target nucleic acid. 2. The microfluidic cartridge of embodiment 1, wherein the sample metering channel controls volumes of liquids dispensed in a channel or chamber. 3. The microfluidic cartridge of embodiment 2, wherein the sample metering channel is fluidically connected to the detection chamber. 4. The resistance channel of any one of embodiments 1-3, wherein the resistance channel has a serpentine path, an angular path, or a circuitous path. 5. The microfluidic cartridge of any one of embodiments 1-4, wherein the valve is a rotary valve, pneumatic valve, a hydraulic valve, an elastomeric valve. 6. The microfluidic cartridge of any one of embodiments 1-5, wherein the resistance channel is fluidically connected with the valve. 7. The microfluidic cartridge of any one of embodiments 1-6, wherein the valve comprises casing, comprising a “substrate” or an “over-mold.” 8. The microfluidic cartridge of any one of embodiments 1-7, wherein the valve is actuated by a solenoid. 9. The microfluidic cartridge of any one of embodiments 1-8, wherein the valve is controlled manually, magnetically, electrically, thermally, by a bistable circuit, with a piezoelectric material, electrochemically, with phase change, rehologically, pneumatically, with a check valve, with capillarity, or any combination thereof 10. The microfluidic cartridge of any one of embodiments 5-9, wherein the rotary valve fluidically connects at least 3, at least, 4, or at least 5 chambers. 11. The microfluidic cartridge of any one of embodiments 1-10, further comprising an amplification reagent chamber fluidically connected to the amplification chamber. 12. The microfluidic cartridge of embodiment 11, further comprising a sample chamber fluidically connected to the amplification reagent chamber. 13. The microfluidic cartridge of embodiment 12, further comprising a sample inlet connected to the sample chamber. 14. The microfluidic cartridge of embodiment 13, wherein the sample inlet is sealable. 15. The microfluidic cartridge of embodiment 14, wherein the sample inlet forms a seal around the sample. 16. The microfluidic cartridge of any one of embodiments 12-15, wherein the sample chamber comprises a lysis buffer. 17. The microfluidic cartridge of any one of embodiments 12-16, further comprising a lysis buffer storage chamber fluidically connected to the sample chamber. 18. The microfluidic cartridge of embodiment 17, wherein the lysis buffer storage chamber comprises a lysis buffer. 19. The microfluidic cartridge of any one of embodiments 16-18, wherein the lysis buffer is a dual lysis/amplification buffer. 20. The microfluidic cartridge of any one of embodiments 17-19, wherein the lysis buffer storage chamber is fluidically connected to the sample chamber through a second valve. 21. The microfluidic cartridge of any one of embodiments 12-20, wherein the sample chamber is fluidically connected to the amplification chamber through the amplification reagent chamber. 22. The microfluidic cartridge any one of embodiments 12-20, wherein the sample chamber is fluidically connected to the amplification reagent chamber through the amplification chamber. 23. The microfluidic cartridge of any one of embodiments 11-22, wherein the microfluidic cartridge is configured to direct fluid bidirectionally between the amplification reagent chamber and amplification chamber. 24. The microfluidic cartridge of any one of embodiments 1-23, wherein the detection reagent chamber is fluidically connected to the amplification chamber. 25. The microfluidic cartridge of any one of embodiments 1-24, wherein the amplification chamber is fluidically connected to the detection chamber through the detection reagent chamber. 26. The microfluidic cartridge of any one of embodiments 1-25, further comprising a reagent port above the detection chamber configured to deliver fluid from the detection reagent chamber to the detection chamber. 27. The microfluidic cartridge of any one of embodiments 1-26, wherein the amplification chamber is fluidically connected to the detection reagent chamber through the detection chamber. 28. The microfluidic cartridge of any one of embodiments 1-27, wherein the resistance channel is configured to reduce backflow into the detection chamber and the detection reagent chamber. 29. The microfluidic cartridge of any one of embodiments 2-27, wherein the sample metering channel is configured to direct a predetermined volume of fluid from the detection reagent chamber to the detection chamber. 30. The microfluidic cartridge of any one of embodiments 1-29, wherein the amplification chamber and detection chamber are thermally isolated. 31. The microfluidic cartridge of any one of embodiments 1-30, wherein the detection reagent chamber is fluidically connected to the detection chamber. 32. The microfluidic cartridge of any one of embodiments 1-31, wherein the detection reagent chamber is fluidically connected to the detection chamber via a second resistance channel. 33. The microfluidic cartridge of any one of embodiments 1-32, wherein the resistance channel or the second resistance channel is a serpentine resistance channel. 34. The microfluidic cartridge of any one of embodiments 1-33, wherein the resistance channel or the second resistance channel comprises at least two hairpins. 35. The microfluidic cartridge of any one of embodiments 1-34, wherein the resistance channel or the second resistance channel comprises at least one, at least 2, at least 3, or at least 4 right angles. 36. The microfluidic cartridge of any one of embodiments 1-35, wherein the amplification chamber comprises a sealable sample inlet. 37. The microfluidic cartridge of embodiment 36, wherein the sample inlet is configured to form a seal around a swab. 38. The microfluidic cartridge of any one of embodiments 1-37, wherein microfluidic cartridge is configured to connect to a first pump to pump fluid from the amplification chamber to the detection chamber. 39. The microfluidic cartridge of any one of embodiments 1-38, wherein microfluidic cartridge is configured to connect to a second pump to pump fluid from the detection reagent chamber to the detection chamber. 40. The microfluidic cartridge of any one of embodiments 38-39, wherein first pump or the second pump is a pneumatic pump, a peristaltic pump, a hydraulic pump, or a syringe pump. 41. The microfluidic cartridge of any one of embodiments 1-40, wherein the amplification chamber is fluidically connected to a port configured to receive pneumatic pressure. 42. The microfluidic cartridge of embodiment 41, wherein the amplification chamber is fluidically connected to the port through a channel. 43. The microfluidic cartridge of any one of embodiments 11-42, wherein the amplification reagent chamber is connected to a second port configured to receive pneumatic pressure. 44. The microfluidic cartridge of embodiment 43, wherein the amplification reagent chamber is fluidically connected to the second port through a second channel. 45. The microfluidic cartridge of any one of embodiments 11-44, wherein the microfluidic cartridge is configured to connect to a third pump to pump fluid from the amplification reagent chamber to the amplification chamber. 46. The microfluidic cartridge of embodiment 45, wherein the third pump is a pneumatic pump, a peristaltic pump, a hydraulic pump, or a syringe pump. 47. The microfluidic cartridge of any one of embodiments 1-46, wherein the detection reagent chamber is connected to a port configured to receive pneumatic pressure. 48. The microfluidic cartridge of any one of embodiments 1-47, wherein the detection reagent chamber is fluidically connected to a third port through a third channel. 49. The microfluidic cartridge of any one of embodiments 1-48, wherein the microfluidic cartridge is configured to connect to a fourth pump to pump fluid from the detection reagent chamber to the detection chamber. 50. The microfluidic cartridge of embodiment 49, wherein the fourth pump is a pneumatic pump, a peristaltic pump, a hydraulic pump, or a syringe pump. 51. The microfluidic cartridge of any one of embodiments 1-50, further comprising a plurality of ports configured to couple to a gas manifold, wherein the plurality of ports is configured to receive pneumatic pressure. 52. The microfluidic cartridge of any one of embodiments 1-51, wherein any chamber of the microfluidic cartridge is connected to the plurality of ports of embodiment 50. 53. The microfluidic cartridge of any one of embodiments 1-52, wherein the valve is opened upon application of current electrical signal. 54. The microfluidic cartridge of any one of embodiments 1-53, wherein the detection reagent chamber is circular. 55. The microfluidic cartridge of any one of embodiments 1-53, wherein the detection reagent chamber is elongated. 56. The microfluidic cartridge of any one of embodiments 1-53, wherein the detection reagent chamber is hexagonal. 57. The microfluidic cartridge of any one of embodiments 2-56, wherein a region of the resistance channel is molded to direct flow in a direction perpendicular to the net flow direction. 58. The microfluidic cartridge of any one of embodiments 2-56, wherein a region of the resistance channel is molded to direct flow in a direction perpendicular to the axis defined by two ends of the resistance channel. 59. The microfluidic cartridge of any one of embodiments 2-58, wherein a region of the resistance channel is molded to direct flow along the z-axis of the microfluidic cartridge. 60. The microfluidic cartridge of any one of embodiments 1-59, wherein the valve is fluidically connected to two detection chambers via an amplification mix splitter. 61. The microfluidic cartridge of any one of embodiments 1-60, wherein the valve is fluidically connected to 3, 4, 5, 6, 7, 8, 9, or 10 detection chambers via an amplification mix splitter. 62. The microfluidic cartridge of any one of embodiments 1-61, further comprising a second valve fluidically connected to the detection reagent chamber and the detection chamber. 63. The microfluidic cartridge of any one of embodiments 1-62, wherein the detection chamber is vented with a hydrophobic PTFE vent. 64. The microfluidic cartridge of any one of embodiments 1-63, wherein the detection chamber comprises an optically transparent surface. 65. The microfluidic cartridge of any one of embodiments 1-64, wherein the amplification chamber is configured to hold from 10 μL to 500 μL of fluid. 66. The microfluidic cartridge of any one of embodiments 11-65, wherein the amplification reagent chamber is configured to hold from 10 μL to 500 μL of fluid. 67. The microfluidic cartridge of any one of embodiments 1-66, wherein the microfluidic cartridge is configured to accept from 2 μL to 100 μL of a sample comprising a nucleic acid. 68. The microfluidic cartridge of any one of embodiments 1-67, wherein the amplification reagent chamber comprises between 5 and 200 μl an amplification buffer. 69. The microfluidic cartridge of any one of embodiments 1-68, wherein the amplification chamber comprises 45 μl amplification buffer. 70. The microfluidic cartridge of any one of embodiments 1-69, wherein the detection reagent chamber stores from 5 to 200 μl of fluid containing the programmable nuclease, the guide nucleic acid, and the labeled detector nucleic acid. 71. The microfluidic cartridge of any one of embodiments 1-70, comprising 2, 3, 4, 5, 6, 7, or 8 detection chambers. 72. The microfluidic cartridge of embodiment 71, wherein the 2, 3, 4, 5, 6, 7, or 8 detection chambers are fluidically connected to a single sample chamber. 73. The microfluidic cartridge of any one of embodiments 1-72, wherein the detection chamber holds up to 100 μL, 200 μL, 300 μL, or 400 μL of fluid. 74. The microfluidic cartridge of any one of embodiments 1-73, wherein the microfluidic cartridge comprises 5-7 layers. 75. The microfluidic cartridge of any one of embodiments 1-74, wherein the cartridge comprises layers as shown in
The following embodiments recite non-limiting permutations of combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is contemplated as depending from or relating to every previous or subsequent numbered embodiment, independent of their order as listed. 1. A system for detecting a target nucleic acid, said system comprising: a guide nucleic acid targeting a target sequence from a virus; a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target sequence; and a reporter, wherein the reporter is capable of being cleaved by the activated nuclease, thereby generating a detectable signal. 2. The system of embodiment 1, wherein the reporter comprises a single stranded reporter comprising a detection moiety. 3. The system of embodiment 1, wherein the virus comprises an influenza virus or a coronavirus. 4. The system of embodiment 3, wherein the influenza virus comprises an influenza A virus, influenza B virus, or a combination thereof 5. The system of embodiment 1, wherein the virus comprises a respiratory virus. 6. The system of embodiment 5, wherein the respiratory virus is an upper respiratory virus. 7. The system of embodiment 1, wherein the guide nucleic acid targets a plurality of target sequences. 8. The system of embodiment 1, wherein the system comprises a plurality of guide sequences tiled against the virus. 9. The system of embodiment 7, wherein the plurality of target sequences comprises sequences from influenza A virus, influenza B virus, and a third pathogen. 10. The system of embodiment 2, wherein the single stranded reporter comprises a detection moiety at the 5′ end. 11. The system of embodiment 2, wherein the single stranded reporter comprises a biotin-dT/FAM moiety or a biotin-dT/ROX moiety. 12. The system of embodiment 2, wherein the single stranded reporter comprises a chemical functional handle at the 3′end capable of being conjugated to a substrate. 13. The system of embodiment 12, wherein the substrate is a magnetic bead. 14. The system of embodiment 12, wherein the substrate is a surface of a reaction chamber. 15. The system of embodiment 14, wherein downstream of the reaction chamber is a test line. 16. The system of embodiment 15, wherein the test line comprises a streptavidin. 17. The system of embodiment 15, wherein downstream of the test line is a flow control line. 18. The system of embodiment 17, wherein the flow control line comprises an anti-IgG antibody. 19. The system of embodiment 18, wherein the anti-IgG antibody comprises an anti-rabbit IgG antibody. 20. The system of embodiment 11, wherein the activated nuclease is capable of cleaving the single stranded reporter and releases the biotin-dT/FAM moiety or the biotin-dT/ROX moiety. 21. The system of embodiment 20, wherein the biotin-dT/FAM moiety is capable of binding the streptavidin at the test line. 22. The system of embodiment 1, wherein the reporter is an electroactive reporter. 23. The system of embodiment 22, wherein the electroactive reporter comprises biotin and methylene blue. 24. The system of embodiment 1, wherein the reporter is an enzyme-nucleic acid. 25. The system of embodiment 24, wherein the enzyme-nucleic acid is an invertase enzyme. 26. The system of embodiment 24, wherein an enzyme of the enzyme-nucleic acid is a sterically hindered enzyme. 27. The system of embodiment 24, wherein upon cleavage of a nucleic acid of the enzyme-nucleic acid, the enzyme is functional. 28. The system of embodiment 1, wherein the detectable signal is a colorimetric signal, a fluorescence signal, an amperometric signal, or a potentiometric signal. 29. A method of detecting a target nucleic acid in a sample comprising: contacting the sample with a guide nucleic acid targeting a target sequence; a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target sequence; and a reporter, wherein the reporter is capable of being cleaved by the activated nuclease, thereby generating a detectable signal. 30. The method of embodiment 29, wherein the target nucleic acid is from an exogenous pathogen. 31. The method of embodiment 30, wherein the exogenous pathogen comprises a virus. 32. The method of embodiment 31, wherein the virus comprises an influenza virus or a coronavirus. 33. The method of embodiment 32, wherein the influenza virus comprises an influenza A virus, influenza B virus, or a combination thereof 34. The method of embodiment 31, wherein the virus comprises a respiratory virus. 35. The method of embodiment 34, wherein the respiratory virus is an upper respiratory virus. 36. The method of embodiment 31, wherein the detectable signal indicates presence of the virus in the sample. 37. The method of embodiment 31, wherein the method further comprises diagnosing a subject from which the sample was taken with the virus. 38. The method of embodiment 37, wherein the subject is a human. 39. The method of embodiment 29, wherein the sample is a buccal swab, a nasal swab, or urine. 40. The method of embodiment 29, wherein the reporter comprises a single stranded reporter comprising a detection moiety. 41. The method of embodiment 29, wherein the guide nucleic acid targets a plurality of target sequences. 42. The system of embodiment 31, wherein the method comprises tiling a plurality of guide sequences against the virus. 43. The method of embodiment 42, wherein the plurality of target sequences comprises sequences from influenza A virus, influenza B virus, and a third pathogen. 44. The method of embodiment 40, wherein the single stranded reporter comprises a detection moiety at the 5′ end. 45. The method of embodiment 40, wherein the single stranded reporter comprises a biotin-dT/FAM moiety or a biotin-dT/ROX moiety. 46. The method of embodiment 40, wherein the single stranded reporter comprises a chemical functional handle at the 3′ end capable of being conjugated to a substrate. 47. The method of embodiment 46, wherein the substrate is a magnetic bead. 48. The method of embodiment 46, wherein the substrate is a surface of a reaction chamber. 49. The method of embodiment 48, wherein downstream of the reaction chamber is a test line. 50. The method of embodiment 49, wherein the test line comprises a streptavidin. 51. The method of embodiment 49, wherein downstream of the test line is a flow control line. 52. The method of embodiment 51, wherein the flow control line comprises an anti-IgG antibody. 53. The method of embodiment 52, wherein the anti-IgG antibody comprises an anti-rabbit IgG antibody. 54. The method of embodiment 45, wherein the activated nuclease is capable of cleaving the single stranded reporter and releases the biotin-dT/FAM moiety or the biotin-dT/ROX moiety. 55. The method of embodiment 54, wherein the biotin-dT/FAM moiety is capable of binding the streptavidin at the test line. 56. The method of embodiment 29, wherein the reporter is an electroactive reporter. 57. The method of embodiment 56, wherein the electroactive reporter comprises biotin and methylene blue. 58. The method of embodiment 29, wherein the reporter is an enzyme-nucleic acid. 59. The method of embodiment 58, wherein the enzyme-nucleic acid is an invertase enzyme. 60. The method of embodiment 58, wherein an enzyme of the enzyme-nucleic acid is a sterically hindered enzyme. 61. The method of embodiment 58, wherein upon cleavage of a nucleic acid of the enzyme-nucleic acid, the enzyme is functional. 62. The method of embodiment 29, wherein the detectable signal is a colorimetric signal, a fluorescence signal, an amperometric signal, or a potentiometric signal. 63. The system of embodiment 5, wherein the respiratory virus is a lower respiratory virus. 64. The method of embodiment 34, wherein the respiratory virus is a lower respiratory virus. 65. A composition comprising: a) a DNA-activated programmable RNA nuclease; and b) a guide nucleic acid comprising a segment that is reverse complementary to a segment of a target deoxyribonucleic acid, wherein the DNA-activated programmable RNA nuclease binds to the guide nucleic acid to form a complex. 66. The composition of embodiment 65, further comprising an RNA reporter. 67. The composition of any one of embodiments 65 and 66, further comprising the target deoxyribonucleic acid from a virus. 68. The composition of any one of embodiments 65-67, wherein the target deoxyribonucleic acid is an amplicon of a nucleic acid. 69. The composition of embodiment 68, wherein the nucleic acid is a deoxyribonucleic acid or a ribonucleic acid. 70. The composition of any one of embodiments 65-69, wherein the DNA-activated programmable RNA nuclease is a Type VI CRISPR/Cas enzyme. 71. The composition of any one of embodiments 65-70, wherein the DNA-activated programmable RNA nuclease is a Cas13. 72. The composition of any one of embodiments 65-71, wherein the DNA-activated programmable RNA nuclease is a Cas13a. 73. The composition of embodiment 72, wherein the Cas13a is LbuCas13a or LwaCas13a. 74. The composition of any one of embodiments 65-73, wherein the composition has a pH from pH 6.8 to pH 8.2. 75. The composition of any one of embodiments 65-74, wherein the target deoxyribonucleic acid lacks a guanine at the 3′ end. 76. The composition of any one of embodiments 65-75, wherein the target deoxyribonucleic acid is a single-stranded deoxyribonucleic acid. 77. The composition of any one of embodiments 65-76, further comprising a support medium. 78. The composition of any one of embodiments 65-77, further comprising a lateral flow assay device. 79. The composition of any one of embodiments 65-78, further comprising a device configured for fluorescence detection. 80. The composition of any one of embodiments 65-79, further comprising a second guide nucleic acid and a DNA-activated programmable DNA nuclease, wherein the second guide nucleic acid comprises a segment that is reverse complementary to a segment of a second target deoxyribonucleic acid comprising a guide nucleic acid. 81. The composition of embodiment 80, further comprising a DNA reporter. 82. The composition of any one of embodiments 80 and 81, wherein the DNA-activated programmable DNA nuclease is a Type V CRISPR/Cas enzyme. 83. The composition of any one of embodiments 81-82, wherein the DNA-activated programmable DNA nuclease is a Cas12. 84. The composition of embodiment 83, wherein the Cas12 is a Cas12a, Cas12b, Cas12c, Cas12d, or Cas12e. 85. The composition of any one of embodiments 80-82, wherein the DNA-activated programmable DNA nuclease is a Cas14. 86. The composition of embodiment 85, wherein the Cas14 is a Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas 14f, Cas14g, or Cas14h. 87. The composition of embodiment 82, wherein the type V CRIPSR/Cas effector protein is a Case protein. 88. The composition of embodiment 87, wherein the Case protein has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 274-SEQ ID NO: 321. 89. The composition of any one of embodiments 87-88, wherein the Case protein is selected from SEQ ID NO: 274-SEQ ID NO: 321. 90. A method of assaying for a target deoxyribonucleic acid from a virus in a sample, the method comprising: contacting the sample to a complex comprising a guide nucleic acid and a DNA-activated programmable RNA nuclease, wherein the guide nucleic acid comprises a segment that is reverse complementary to a segment of the target deoxyribonucleic acid, and assaying for a signal produced by cleavage of at least some RNA reporters of a plurality of RNA reporters. 91. A method of assaying for a target ribonucleic acid from a virus in a sample, the method comprising: amplifying a nucleic acid in a sample to produce a target deoxyribonucleic acid; contacting the target deoxyribonucleic acid to a complex comprising a guide nucleic acid and a DNA-activated programmable RNA nuclease, wherein the guide nucleic acid comprises a segment that is reverse complementary to a segment of the target deoxyribonucleic acid, and assaying for a signal produced by cleavage of at least some RNA reporters of a plurality of RNA reporters. 92. The method of any one of embodiments 90 or 91, wherein the DNA-activated programmable RNA nuclease is a Type VI CRISPR nuclease. 93. The method of any one of embodiments 90-92, wherein the DNA-activated programmable RNA nuclease is a Cas13. 94. The method of embodiment 93, wherein the Cas13 is a Cas13a. 95. The method of embodiment 94, wherein the Cas13a is LbuCas13a or LwaCas13a. 96. The method of any one of embodiments 90-95, wherein cleavage of the at least some RNA reporters of the plurality of reporters occurs from pH 6.8 to pH 8.2. 97. The method of any one of embodiments 90-96, wherein the target deoxyribonucleic acid lacks a guanine at the 3′ end. 98. The method of any one of embodiments 90-97, wherein the target deoxyribonucleic acid is a single-stranded deoxyribonucleic acid. 99. The method of any one of embodiments 90-98, wherein the target deoxyribonucleic acid is an amplicon of a ribonucleic acid. 100. The method of any one of embodiments 90-99, wherein the target deoxyribonucleic acid or the ribonucleic acid is from an organism. 101. The method of embodiment 100, wherein the organism is a virus, bacteria, plant, or animal. 102. The method of any one of embodiments 90-101, wherein the target deoxyribonucleic acid is produced by a nucleic acid amplification method. 103. The method of embodiment 102, wherein the nucleic acid amplification method is isothermal amplification. 104. The method of embodiment 102, wherein the nucleic acid amplification method is thermal amplification. 105. The method of embodiment 102, wherein the nucleic acid amplification method is recombinase polymerase amplification (RPA), transcription mediated amplification (TMA), strand displacement amplification (SDA), helicase dependent amplification (HDA), loop mediated amplification (LAMP), rolling circle amplification (RCA), single primer isothermal amplification (SPIA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), or improved multiple displacement amplification (IMDA), or nucleic acid sequence-based amplification (NASBA). 106. The method of any one of embodiments 90-105, wherein the signal is fluorescence, luminescence, colorimetric, electrochemical, enzymatic, calorimetric, optical, amperometric, or potentiometric. 107. The method of any one of embodiments 90-106, further comprising contacting the sample to a second guide nucleic acid and a DNA-activated programmable DNA nuclease, wherein the second guide nucleic acid comprises a segment that is reverse complementary to a segment of a second target deoxyribonucleic acid comprising a guide nucleic acid. 108. The method of embodiment 39, further comprising assaying for a signal produced by cleavage of at least some DNA reporters of a plurality of DNA reporters. 109. The method of any one of embodiments 107 and 108, wherein the DNA-activated programmable DNA nuclease is a Type V CRISPR nuclease. 110. The method of any one of embodiments 107-109, wherein the DNA-activated programmable DNA nuclease is a Cas12. 111. The method of embodiment 110, wherein the Cas12 is a Cas12a, Cas12b, Cas12c, Cas12d, or Cas12e. 112. The method of embodiments 107-109, wherein the DNA-activated programmable DNA nuclease is a Cas14. 113. The method of embodiment 112, wherein the Cas14 is a Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, or Cas14h. 114. The method of embodiment 107-109, wherein the DNA-activated programmable DNA nuclease is a Case protein. 115. The method of embodiment 114, wherein the Case protein has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to any one of SEQ ID NO: 274-SEQ ID NO: 321. 116. The method of any one of embodiments 114-115, wherein the CasΦ protein is selected from SEQ ID NO: 274-SEQ ID NO: 321. 117. The method of any one of embodiments 90-116, wherein the guide nucleic acid comprises a crRNA. 118. The method of any one of embodiments 90-117, wherein the guide nucleic acid comprises a crRNA and a tracrRNA. 119. The method of any one of embodiments 90-118, wherein the signal is present prior to cleavage of the at least some RNA reporters. 120. The method of any one of embodiments 90-119, wherein the signal is absent prior to cleavage of the at least some RNA reporters. 121. The method of any one of embodiments 90-120, wherein the sample comprises blood, serum, plasma, saliva, urine, mucosal sample, peritoneal sample, cerebrospinal fluid, gastric secretions, nasal secretions, sputum, pharyngeal exudates, urethral or vaginal secretions, an exudate, an effusion, or tissue. 122. The method of any one of embodiments 90-121, wherein the method is carried out on a support medium. 123. The method of any one of embodiments 90-122, wherein the method is carried out on a lateral flow assay device. 124. The method of any one of embodiments 90-123, wherein the method is carried out on a device configured for fluorescence detection. 125. A method of designing a plurality of primers for amplification of a target nucleic acid, the method comprising: providing a target nucleic acid, wherein a guide nucleic acid hybridizes to the target nucleic acid and wherein at least 60% of a sequence of the target nucleic acid is between an F1c region and a B1 region or between an F1 and a B1c region; and designing the plurality of primers comprising: i) a forward inner primer comprising a sequence of the F1c region 5′ of a sequence of an F2 region; ii) a backward inner primer comprising a sequence of the B1c region 5′ of a sequence of a B2 region; iii) a forward outer primer comprising a sequence of an F3 region; and iv) a backward outer primer comprising a sequence of a B3 region. 126. A method of detecting a target nucleic acid in a sample, the method comprising: contacting the sample to: a plurality of primers comprising: i) a forward inner primer comprising a sequence corresponding to an F1c region 5′ of a sequence corresponding to an F2 region; ii) a backward inner primer comprising a sequence corresponding to a B1 c region 5′ of a sequence corresponding to a B2 region; iii) a forward outer primer comprising a sequence corresponding to an F3 region; and iv) a backward outer primer comprising a sequence corresponding to a B3 region; a guide nucleic acid, wherein the guide nucleic acid hybridizes to the target nucleic acid and wherein at least 60% of a sequence of the target nucleic acid is between the F1c region and a B1 region or between an F1 region and the B1c region; a reporter; and a programmable nuclease that cleaves the reporter when complexed with the guide nucleic acid; and measuring a detectable signal produced by cleavage of the reporter, wherein the measuring provides for detection of the target nucleic acid in the sample. 127. The method of any one of embodiments 125-126, wherein the sequence between the F1c region and the B1 region or the sequence between the B1c region and the F1 region is at least 50% reverse complementary to the guide nucleic acid sequence. 128. The method of any one of embodiments 125-127, wherein the guide nucleic acid sequence is reverse complementary to no more than 50% of the forward inner primer, the backward inner primer, or a combination thereof 129. The method of any one of embodiments 125-128, wherein the guide nucleic acid does not hybridize to the forward inner primer and the backward inner primer. 130. The method of any one of embodiments 125-129, wherein a protospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of the target nucleic acid. 131. The method of any one of embodiments 125-130, wherein a protospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of the B1 region and 5′ of the F1c region or the protospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of the F1 region and 5′ of the B1c region. 132. The method of any one of claims 125-131, wherein the 3′ end of the target nucleic acid is 5′ of the 5′ end of the F3c region or the 3′ end of the target nucleic acid is 5′ of the 5′ end of the B3c region. 133. The method of any one of embodiments 125-132, wherein the 3′ end of the target nucleic acid is 5′ of the 5′ end of the F2c region or 3′ end of the target nucleic acid is 5′ of the 5′ end of the B2c region. 134. The method of any one of embodiments 125-133, wherein the target nucleic acid is between the F1c region and the B1 region and the 3′ end of the target nucleic acid is 5′ of the 3′ end of the F2c region, or wherein the target nucleic acid is between the B1c region and the F1 region and the 3′ end of the target nucleic acid is 5′ of the 3′ end of the B2c region. 135. The method of any one of embodiments 125-134, wherein the guide nucleic acid has a sequence reverse complementary to no more than 50% of the forward inner primer, the backward inner primer, the forward outer primer, the backward outer primer, or any combination thereof 136. The method of any one of embodiments 125-135, wherein the guide nucleic acid sequence does not hybridize to the forward inner primer, the backward inner primer, the forward outer primer, the backward outer primer, or any combination thereof 137. The method of any one of embodiments 125-136, wherein the guide nucleic acid sequence has a sequence reverse complementary to no more than 50% of a sequence of an F3c region, an F2c region, the F1c region, the B1c region, an B2c region, an B3c region, or any combination thereof 138. The method of any one of embodiments 125-137, wherein the guide nucleic acid sequence does not hybridize to a sequence of an F3c region, an F2c region, the F1c region, the B1c region, an B2c region, an B3c region, or any combination thereof 139. A method of designing a plurality of primer for amplification of a target nucleic acid, the method comprising: providing the target nucleic acid comprising a sequence between a B2 region and a B1 region or between an F2 region and an F1 region that hybridizes to a guide nucleic acid; and designing the plurality of primers comprising: i) a forward inner primer comprising a sequence of the F1c region 5′ of a sequence of an F2 region; ii) a backward inner primer comprising a sequence of the B1c region 5′ of a sequence of a B2 region; iii) a forward outer primer comprising a sequence of an F3 region; and iv) a backward outer primer comprising a sequence of a B3 region. 140. A method of designing a plurality of primer for amplification of a target nucleic acid, the method comprising: providing the target nucleic acid comprising a sequence between a F1c region and an F2c region or between a B1c region and a B2c region that hybridizes to a guide nucleic acid; and designing the plurality of primers comprising: i) a forward inner primer comprising a sequence of the F1c region 5′ of a sequence of an F2 region; ii) a backward inner primer comprising a sequence of the B1c region 5′ of a sequence of a B2 region; iii) a forward outer primer comprising a sequence of an F3 region; and iv) a backward outer primer comprising a sequence of a B3 region. 141. A method of detecting a target nucleic acid in a sample, the method comprising: contacting the sample to: a plurality of primers comprising: i) a forward inner primer comprising a sequence corresponding to an F 1 c region 5′ of a sequence corresponding to an F2 region; ii) a backward inner primer comprising a sequence corresponding to a B1c region 5′ of a sequence corresponding to a B2 region; iii) a forward outer primer comprising a sequence corresponding to an F3 region; and iv) a backward outer primer comprising a sequence corresponding to a B3 region; a guide nucleic acid, wherein the target nucleic acid comprises a sequence between a B2 region and a B1 region or between the F2 region and an F1 region that hybridizes to the guide nucleic acid; a reporter; and a programmable nuclease that cleaves the reporter when complexed with the guide nucleic acid; and measuring a detectable signal produced by cleavage of the reporter, wherein the measuring provides for detection of the target nucleic acid in the sample. 142. A method of detecting a target nucleic acid in a sample, the method comprising: contacting the sample to: a plurality of primers comprising: i) a forward inner primer comprising a sequence corresponding to an F1c region 5′ of a sequence corresponding to an F2 region; ii) a backward inner primer comprising a sequence corresponding to a B1c region 5′ of a sequence corresponding to a B2 region; iii) a forward outer primer comprising a sequence corresponding to an F3 region; and iv) a backward outer primer comprising a sequence corresponding to a B3 region; a guide nucleic acid, wherein the target nucleic acid comprises a sequence between the F1c region and an F2c region or between the B1c region and a B2c region that hybridizes to the guide nucleic acid; a reporter; and a programmable nuclease that cleaves the reporter when complexed with the guide nucleic acid; and measuring a detectable signal produced by cleavage of the reporter, wherein the measuring provides for detection of the target nucleic acid in the sample. 143. The method of any one embodiment 139 or embodiment 141, wherein a protospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of the B2 region and 5′ of the B1 region or the protospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of the F2 region and 5′ of the Fl region. 144. The method of any one embodiment 140 or embodiment 142, wherein a protospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of the B1c region and 5′ of the B2c region or the protospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of the F1c region and 5′ of the F2c region. 145. The method of any one of embodiments 139-144, wherein a protospacer adjacent motif (PAM) or a protospacer flanking site (PFS) is 3′ of the target nucleic acid. 146. The method of embodiment 145, wherein the PAM and the PFS are 5′ of the 5′ end of the F1c region, 5′ of the 5′ end of the B1c region, 3′ of the 3′ end of the F3 region, 3′ of the 3′ end of the B3 region, 3′ of the 3′ end of the F2 region, 3′ of the 3′ end of the B2 region, or any combination thereof 147. The method of embodiment 146, wherein the PAM and the PFS do not overlap the F2 region, the B3 region, the F1c region, the F2 region, the B1c region, the B2 region, or any combination thereof 148. The method of any one of embodiments 145-147, wherein the PAM and the PFS do not hybridize to the forward inner primer, the backward inner primer, the forward outer primer, the backward outer primer, or any combination thereof 149. The method of any one of embodiments 125-148, wherein the plurality of primers further comprises a loop forward primer. 150. The method of any one of embodiments 125-149, wherein the plurality of primers further comprises a loop backward primer. 151. The method of any one of embodiments 149-150, wherein the loop forward primer is between an F lc region and an F2c region. 152. The method of any one of embodiments 150-151, wherein the loop backward primer is between a B1 c region and a B2c region. 153. The method of any one of embodiments 125-152, wherein the target nucleic acid comprises a single nucleotide polymorphism (SNP). 154. The method of embodiment 153, wherein the single nucleotide polymorphism (SNP) comprises a HERC2 SNP. 155. The method of embodiment 153, wherein the single nucleotide polymorphism (SNP) is associated with an increased risk or decreased risk of cancer. 156. The method of any one of embodiments 126-138 or 141-155, wherein the target nucleic acid comprises a single nucleotide polymorphism (SNP), and wherein the detectable signal is higher in the presence of a guide nucleic acid that is 100% complementary to the target nucleic acid comprising the single nucleotide polymorphism (SNP) than in the presence of a guide nucleic acid that is less than 100% complementary to the target nucleic acid comprising the single nucleotide polymorphism (SNP). 157. The method of any one of embodiments 125-156, wherein the plurality of primers and the guide nucleic acid are present together in a sample comprising the target nucleic acid. 158. The method of any one of embodiments 126-138 or 141-157, wherein the contacting the sample to the plurality of primers results in amplifying the target nucleic acid. 159. The method of embodiment 158, wherein the amplifying and the contacting the sample to the guide nucleic acid occurs at the same time. 160. The method of embodiment 158, wherein the amplifying and the contacting the sample to the guide nucleic acid occur at different times. 161. The method of any one of embodiments 126-138 or 141-160, wherein the method further comprises providing a polymerase, a dATP, a dTTP, a dGTP, a dCTP, or any combination thereof 162. The method of any one of embodiments 125-161, wherein the target nucleic acid is from a virus. 163. The method of embodiment 162, wherein the virus comprises an influenza virus, respiratory syncytial virus, coronavirus, or a combination thereof 164. The method of embodiment 163, wherein the influenza virus comprises an influenza A virus, influenza B virus, or a combination thereof 165. The method of any one of embodiments 162-164, wherein the virus comprises a respiratory virus. 166. The method of embodiment 165, wherein the respiratory virus is an upper respiratory virus. 167. The system of any one of embodiments 1-28, wherein the system further comprises a forward inner primer, a backward inner primer, a forward outer primer, a backward outer primer, a loop forward primer, a loop backward primer, or any combination thereof 168. The method of any one of embodiments 29-64, the method further comprising contacting the sample with a forward inner primer, a backward inner primer, a forward outer primer, a backward outer primer, a loop forward primer, a loop backward primer, or any combination thereof 169. The method of embodiment 87, the method further comprising amplifying the target deoxyribonucleic acid with a forward inner primer, a backward inner primer, a forward outer primer, a backward outer primer, a loop forward primer, a loop backward primer, or any combination thereof 170. The method of any one of embodiments 88-125, wherein the amplifying comprises contacting the sample to a forward inner primer, a backward inner primer, a forward outer primer, a backward outer primer, a loop forward primer, a loop backward primer, or any combination thereof 171. The system or method of any one of embodiments 3, 32, or 163, wherein the coronavirus is SARS CoV-2. 172. The method of embodiment 105, wherein the nucleic acid amplification method is loop mediated amplification (LAMP).
The following examples are illustrative and non-limiting to the scope of the devices, systems, fluidic devices, kits, and methods described herein.
A biological sample from an individual can be tested to determine whether the individual has influenza. The biological sample can be tested to detect the presence or absence of a target nucleic acid indicative of a influenza A or influenza B virus.
An individual obtains a biological sample of urine and applies the biological sample to the reagents described herein in a reagent chamber provided in a kit. The reagents comprise a guide nucleic acid targeting a nucleic acid present in and specific to the virus, a programmable nuclease, and a single stranded detector nucleic acid with a detection moiety. The biological sample has the influenza virus, and the target nucleic acid from the virus binds to the guide nucleic acid and activates the programmable nuclease to cleave the target nucleic acid and the single stranded detector nucleic acid.
After the sample and the reagents are contacted for a predetermined time, the individual can apply the reacted sample and reagents to a sample pad region on a support medium. The support medium comprises a lateral flow assay test strip encased in a protective housing with openings for the sample pad region to apply the reacted sample and reagents and for a detection region for reading the test results. The housing also has fiduciary markers, a reference color scale, and a barcode that identifies the test performed by the kit. As the reacted sample and reagents move along the lateral flow assay test strip to the detection region, the detection moiety from the cleaved single stranded detector nucleic acid binds with a capture molecule on the support medium and a detection molecule in a detection region to generate a detectable signal on the support medium. The detectable signal can be line in the detection region of the support medium. Once the test is complete, a line for a positive control marker and another line for a positive test become visible through the detection region opening.
After a predetermined amount of time after applying the reacted sample and reagents to the support medium, the individual can use a mobile device to obtain the test results. The individual can open a mobile application for reading of the test results on a mobile device with a camera and take an image of the support medium, including the detection region, barcode, reference color scale, and fiduciary markers on the housing, using the camera of the mobile device and the GUI of the mobile application. The mobile application identifies the test based on the barcode in the image, analyzes the detection region in the image with the fiduciary markers and a reference color scale on the housing in the same image based on the identification of the test with the barcode, and determines the presence or absence of the virus. The mobile application can present the results of the test to the individual. The mobile application can store the test results in the mobile application. The mobile application can communicate with a remote device and transfer the data of the test results. The test results can be viewable remotely from the remote device by another individual, including a healthcare professional.
A biological sample from an individual can be tested to determine whether the individual has influenza. The biological sample can be tested to detect the presence or absence of a target nucleic acid indicative of influenza A or influenza B.
An individual obtains a biological sample of urine and applies the biological sample to the reagents described herein in a reagent chamber provided in a kit. The reagents comprise a guide nucleic acid targeting a nucleic acid present in and specific to the virus, a programmable nuclease, and a single stranded detector nucleic acid.
After the sample and the reagents are contacted for a predetermined time, the individual can place one end of a support medium into the reagent chamber to apply the reacted sample and reagents to a sample pad region on the support medium. The support medium comprises a lateral flow assay test strip. As the reacted sample and reagents move along the test strip to the detection region, a line for a positive control marker becomes visible in the detection region. After a predetermined amount of time after applying the reacted sample and reagents to the support medium, the support medium can be placed into a protective housing with an opening for the detection region for reading the test results, fiduciary markers, a reference color scale, and a barcode that identifies the test performed by the kit.
The individual can use a mobile device to obtain the test results. The individual can open a mobile application for reading of the test results on a mobile device with a camera and take an image of the support medium, including the detection region, barcode, reference color scale, and fiduciary markers on the housing, using the camera of the mobile device and the mobile application. The mobile application identifies the test based on the barcode in the image, analyzes the detection region in the image with the fiduciary markers and a reference color scale on the housing in the same image based on the identification of the test with the barcode, and determines the presence or absence of the virus. The mobile application can present the results of the test to the individual.
A biological sample from an individual can be tested to determine whether the individual has influenza. The biological sample can be tested to detect the presence or absence of a target nucleic acid indicative of influenza A or influenza B virus.
An individual obtains a biological sample of urine and applies the biological sample to the reagents described herein on a sample pad region on a support medium provided in a kit. The reagents comprise a guide nucleic acid targeting a nucleic acid present in and specific to the virus, a programmable nuclease, and a single stranded detector nucleic acid. The support medium comprises a lateral flow assay test strip encased in a protective housing with an opening for the detection region for reading the test results, fiduciary markers, a reference color scale, and a barcode that identifies the test performed by the kit.
After the sample and the reagents are contacted for a predetermined time on the support medium, the reacted sample and reagents move along the support medium to a detection region on the support medium. The individual can optionally place a small volume of buffer to help move the reacted sample and reagents to the detection region. As the reacted sample and reagents move along the test strip to the detection region, a line for a positive control marker becomes visible in the detection region.
The individual can use a mobile device to obtain the test results. The individual can use a mobile device to obtain the test results. The mobile application identifies the test based on the barcode in the image, analyzes the detection region in the image, and determines the presence or absence of the virus. The mobile application can present the results of the test to the individual.
A biological sample from an individual can be tested to determine whether the individual has one or more than one strains of influenza (e.g., influenza A, influenza B. The biological sample can be tested to detect the presence or absence of one or more of target nucleic acids, where the individual target nucleic acid is indicative of a virus.
An individual obtains a biological sample of urine and applies the biological sample to multiple reagent chambers provided in a kit to test for a panel of influenza virus strains. Each reagents chamber comprise the reagents specific to detect one influenza virus strain. The reagents in each reagent chamber comprise a guide nucleic acid targeting a nucleic acid present in the virus; a programmable nuclease; and a single stranded detector nucleic acid.
After the sample and the reagents are contacted for a predetermined time, the individual can apply the reacted sample and reagents from one of the reagent chambers to a matched sample pad region on a support medium. Each reagent chamber has a matching sample pad region on the support medium. The support medium comprises multiple lateral flow assay test strips encased in a protective housing with openings for the matched sample pad regions to apply the reacted sample and reagents from the matching reagent chamber and for a detection region for reading the test results. The housing also has fiduciary markers, a reference color scale, and a barcode that identifies the tests performed by the kit. As the reacted sample and reagents move along the lateral flow assay test strip to the detection region, a positive control marker for each lateral flow test strip becomes visible through the detection region opening.
After a predetermined amount of time after applying the reacted sample and reagents to the support medium, the individual can use a mobile device to obtain the test results. The individual can use a mobile device to obtain the test results. The individual can use a mobile device to obtain the test results. The mobile application identifies the test based on the barcode in the image, analyzes the detection region in the image, and determines the presence or absence of the virus (influenza A, influenza B, or influenza A and B). The mobile application can present the results of the test to the individual.
A biological sample from an individual can be tested to determine whether the individual has one or more than one strains of influenza (e.g., influenza A, influenza B. The biological sample can be tested to detect the presence or absence of one or more of target nucleic acids, where the individual target nucleic acid is indicative of a virus.
An individual obtains a biological sample of urine and applies the biological sample to a reagent chamber provided in a kit to test for a panel of influenza virus strains. The reagents chamber comprises multiple sets of reagents to detect multiple influenza virus strains. One set of reagents to detect one influenza strain comprise a guide nucleic acid targeting a nucleic acid present in and specific to the virus; a programmable nuclease; and a single stranded detector nucleic acid.
After the sample and the reagents are contacted for a predetermined time, the individual can apply the reacted sample and reagents to a sample pad region on a support medium. The support medium comprises a multiplexed lateral flow assay test strip that can detect multiple detector molecules on the test strip. The lateral flow assay strip encased in a protective housing with openings for the sample pad region to apply the reacted sample and reagents and for a detection region for reading the test results. The housing also has fiduciary markers, a reference color scale, and a barcode that identifies the tests performed by the kit. As the reacted sample and reagents move along the lateral flow assay test strip to the detection region, a positive control marker for each lateral flow test strip becomes visible through the detection region opening.
After a predetermined amount of time after applying the reacted sample and reagents to the support medium, the individual can use a mobile device to obtain the test results. The individual can use a mobile device to obtain the test results. The individual can use a mobile device to obtain the test results. The mobile application identifies the test based on the barcode in the image, analyzes the detection region in the image, and determines the presence or absence of the virus. The mobile application can present the results of the test to the individual.
This example illustrates detection of a nucleic acid from a respiratory virus (e.g., an influenza virus) using a fluidic device. A CRISPR-Cas reaction for detection of a target nucleic acid from a respiratory virus (e.g., an influenza virus) in a sample is carried out using a fluidic device.
The fluidic device is one of the three fluidic devices of
A sample containing the target nucleic acid of interest from a respiratory virus (e.g., an influenza virus) is introduced into a fluidic device of
This example describes electrochemical detection of target nucleic acids from a respiratory virus (e.g., an influenza virus) in DETECTR reactions using CRISPR/Cas systems. In this assay a biotin-streptavidin signal enhancement method is employed using a biotinylated CRISPR-Cas reporter molecule, which is cleaved by the enzyme in the presence of a positive DETECTR reaction (one in which the target nucleic acid is present).
Electrochemical detection is tested as an alternative to (1) use of ferrocene-labelled oligos immobilized on the electrode surface and (2) coupling of DETECTR to an invertase catalyzed reaction, also disclosed herein. The latter reaction produces glucose that be detected with a glucometer directly, or indirectly. Electrochemical detection and detection using ferrocene-labelled oligos are both potentiometric, while invertase catalyzed reactions are amperometric.
The reporter is cleaved using a DNAse enzyme and cleavage results in an increase in current at an oxidation peak compared to when the reporter was intact. Results are collected using a benchtop, gold-standard electrochemical analyzer (uSTAT, Metrohm, USA). The sequence of the reporter is /5Biosg/TTTTTTTTTTTTTTTTTTTT/3MeBlN/ (SEQ ID NO: 373). A cyclic voltammogram is obtained, wherein cleavage of the electroactive reporter leads to an increase in current.
This example describes a fluorescence-based device for detection of target nucleic acids from a respiratory virus (e.g., an influenza virus) in DETECTR reactions using CRISPR-Cas systems. Two approaches are used to develop a miniaturized device for DETECTR reactions for detection of target nucleic acids from a respiratory virus (e.g., an influenza virus). First, a glass capillary (Drummond Scientific, USA) is used as a single, capillarity driven vessel of the DETECTR reaction. Both flash-dried and liquid formulations of the reagents are used. Second, a commercially-available, plastic (TOPAS) microfluidic chip (Microfluidic Chip Shop, Germany) with no mechanical actuation for mixing or reagent delivery is used.
Results are collected using (1) a portable, photodiode-based fluorescence sensor (ESELog, Quiagen Lake Constance, Germany) and (2) a commercially-available transilluminator (E-GEL, Thermofisher, USA). The major advancements of the detection capabilities of this system includes an on-chip DETECR reaction. Real-time measurement of fluorescence from a one-pot reverse transcription-recombinase polymerase amplification-in vitro transcription (RT-RPA-IVT)-DETECTR reaction is carried out on chip
This example describes guide pooling for high sensitivity and broad spectrum detection of target nucleic acids from a respiratory virus (e.g., an influenza virus). While traditional detection requires one gRNA per target sequence/organism, the guide pooling methods disclosed herein, including use of multiple gRNAs with one or more CRISPR effector proteins, is amenable for both improving sensitivity (in the case of guide tiling across a single target sequence/organism) and broad spectrum detection (in the case of detecting multiple target sequences/organisms in a single reaction). Thus, guide pooling is a useful method for enhancing detection sensitivity performance as well as functioning as an initial triage step that is rapid and low-cost (e.g., an “alert” for blood-borne pathogens, pandemic flu, pan-bacterial detection, etc.) as compared to traditional diagnostic methods.
To perform a DETECTR assay, a guide RNA (crRNA) is first complexed to the Cas protein. The complexing reaction is carried out at 37° C. for 30 minutes. Reporter and additional buffer are then added to complete the complex master mix. Finally, the complex is added to the samples to detect sequences specifically targeted by the guide. By pooling multiple guide RNAs designed to target difference sequences or different sequence segments of the same target, it is possible to broaden the detection spectrum in a single reaction and increase the detection efficiency. To achieve this, guide RNAs are individually complexed to Cas protein at high concentration. Multiple guide-protein complex reactions are pooled. After pooling, the reporter and addition buffer are added to complete the pooled complexes for use in the DETECTR assay.
A. Guide Pooling for Detection of Influenza Strains
Methods for guide pooling for detection of influenza strains involved using guide RNAs for different influenza strains (e.g., strains from IAV and/or IBV). Multiple guide RNAs (e.g., 15-20) are designed to target lthe influenza strains.
The programmable nuclease used in the DETECTR assay is LbCas12a (SEQ ID NO: 27). The reporter is an 8-mer ssDNA with a FAM-labeled 5′ end and an Iowa Black FQ-labeled 3′ end.
Guide RNA (crRNA) are individually complexed with LbCas12a protein at high concentration. Each crRNA is mixed with LbCas12a in 1× MBuffer 2 (20 mM Tris HCl, pH8, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol, 50 ug/mL Heparin). The concentrations of the crRNA and protein are at least 4-fold higher than those in the standard single guide complexing reaction. The mixture is incubated at 37° C. for 30 minutes to form the guide-protein complex. TABLE 8 below lists the formulation of the complexing reaction. The volumes are for one DETECTR reaction, and can be scaled up accordingly.
Guide Pooling. At the completion of the incubation, guide pools are generated by combining equal volume of individual guide-protein complexing reactions. Several pools at different n-plex (n=number of different guides) levels are generated.
5 ul of the complexing reaction is used in each 20 ul DETECTR assays. The effective concentration in the assay of guides and protein is one fourth of those in the complexing reactions.
Complex Master Mix. Complex Master Mixes of the guide pools are completed by adding equal volume of Mix2 (containing the ssDNA reporter and additional buffer, the formulation is listed in TABLE 9).
Additionally, Mix2 is added to individual guide-protein complexing reaction to generate single guide complex master mix. In the complex master mix, the concentrations of the guides and proteins are diluted in half
DETECTR assay. The targets are diluted to 200 pM and 20 pM. In each DETECTR assay, 10 ul of complex master mix is mixed with 10 ul of sample in a well of a 384-well plate. The effective concentrations of the guides and protein are one fourth of those in the complexing reaction. The reaction is carried out in a TECAN Infinite 200 pro plate reader at 37 C. The fluorescence raw data file is analyzed using internal software. The kinetics of the DETECTR assay is measured by max rate (estimated rate of cleavage of the reporter by the activated Cas protein). The activity of the guide pools versus the single guide is measured against 200 pM targets (100 pM targets in the final reaction).
Signals are clearly boosted by guide pooling. For example, the signal increases as the n-plex level is increased to 10- and 20-plex and the detection sensitivity is improved from the single guide detection. The guide pools can be adjusted to detect difference targets.
Using guide pooling, detection of 10 pM targets, which is near the detection limit of the single guide assay, is improved and the pooling of the guides improves the sensitivity of the assay.
B. Guide pooling Top-Performing Individual gRNAs to Increase Assay Sensitivity for Detection of RSV
Guide pooling was used to improve the detection limit of an assay for RSV detection. 33 guide RNAs for RSV guides were designed by tiling across the target region. The guide RNAs were screened for activities and top performing guides were selected for pooling. RNA corresponding to the RSV target was generated from in vitro transcription (IVT) reaction. A Cas13a protein was used and the reporter was a 5-mer ssRNA with a 5′ FAM and a 3′ Iowa Black FQ.
This example describes optimization of temperature and temperature tolerance of CRISPR-Cas proteins in CRISPR DETECTR assays for detection of target nucleic acids from respiratory viruses (e.g., influenza virus). The CRISPR diagnostics of the present disclosure leverage the unique biochemical properties of Type V (e.g., Cas12) and Type VI (e.g., Cas13) CRISPR-Cas proteins to enable the specific detection of nucleic acids. These proteins are directed to bind a target nucleic acid from a respiratory virus (e.g., an influenza virus) by a CRISPR RNA (crRNA), which is also known as a guide RNA (gRNA). Once bound to a complementary target sequence, the Cas protein initiates indiscriminate cleavage of surrounding single-strand DNA or single-strand RNA. When coupled to a quenched fluorescence reporter or other cleavage reporter, fluorescent or other signal is generated by the Cas protein only in the presence of the target nucleic acid. CRISPR-Cas proteins are isolated from a variety of natural contexts and therefore have different tolerances for elevated temperatures and optimal temperature ranges. These different tolerances for temperature are used to activate or inhibit the proteins at different stages to allow for other molecular processes, such as target amplification of nucleic acids from respiratory viruses (e.g., an influenza virus), to occur.
In DETECTR assays in which the target nucleic acid is from a respiratory virus (e.g., an influenza virus) and a Cas12 variant (SEQ ID NO: 37) programmable nuclease is used, the Type V protein Cas12 variant has a functional range between 25° C. and 45° C., with maximal activity at 35° C. For the Type V protein LbCas12a (SEQ ID NO: 27), the functional range is from 35° C. to 50° C. with peak activity around 40° C. For the Type VI protein LbuCas13a (SEQ ID NO: 131) the functional range is between 25° C. and 40° C. with maximal activity between 30° C. and 35° C. Type V proteins, such as the Cas12 variant and LbCas12a, are stable and functional at elevated temperatures. In DETECTR assays in which the target nucleic acid is from a respiratory virus (e.g., an influenza virus), the Cas12 variant exhibits activity at a temperature of 37° C. This temperature shifting is exploitable for use in isothermal amplification methods, where the amplification occurs at a higher temperature, but after lowering the reaction temperature the Cas protein is activated without compromising its functionality.
In DETECTR assays in which the target nucleic acid is from a respiratory virus (e.g., an influenza virus), the Cas12 variant is stable after exposure to elevated temperatures for 30 minutes and then lowering the reaction temperature to 37° C. The Cas12 variant is active in 0.5× NEBuffer4 (New England Biolabs)+0.05% Tween and 1× MBuffer3.
This example describes sample preparation protocol and device workflow. Collecting and processing material for diagnostic analysis is typically performed at a point of care facility or a clinical laboratory. There are minimal methods currently available for at home sample collection and nucleic acid extraction for diagnostic analysis. The devices disclosed herein provide an over the counter solution for nucleic acid extraction with or without nucleic acid amplification and with or without the DETECTR reaction. The resulting product from any or all of these modules is applied to a readout device for data collection and subsequent analysis.
A crude sample preparation protocol includes elution of a sample from a sample collection device (e.g. swab) into a buffer that will induce dissociation of the sample into its macromolecule components releasing the genomic nucleic acids. These components include any or all of the following: pH change, chaotropic salts and a detergent (Tween 20, Triton X-100, Deoxycholate, Sodium laurel sulfate or CHAPS. This protocol occurs in a stepwise work flow that would feed into a hand held device. In this device there is at least one chamber that contains the reagents components for the sample preparation protocol.
Examples of crude sample preparation protocols are summarized in TABLE 11.
This example describes methods of isothermal amplification in the CRISPR-Cas diagnostics of the present disclosure, including those diagnostics involving DETECTR assays. CRISPR diagnostics leverage the unique biochemical properties of Type V (e.g. Cas12) and Type VI (e.g. Cas13) CRISPR-Cas proteins to enable the specific detection of nucleic acids. These proteins are directed to their target nucleic acid by a CRISPR RNA (crRNA), which is also known as a guide RNA (gRNA). Once bound to a complementary target sequence, the Cas protein initiates indiscriminate cleavage of surrounding single-strand DNA or single-strand RNA. When coupled to a quenched fluorescence reporter or other cleavage reporter, fluorescent or other signal can be generated by the Cas protein only in the presence of the target nucleic acid. Alone these proteins are capable of detecting in the pM or fM range of target nucleic acid. When coupled to nucleic acid amplification set forth in this example and disclosed elsewhere herein, the sensitivity of CRISPR diagnostics was increased to the aM or zM range. PCR is a commonly used nucleic acid amplification method that generates double stranded DNA (dsDNA) when temperatures are cycled between two or three different temperatures. Nucleic acid amplification methods that function at single temperature are known as isothermal amplification. These methods include LAMP, RPA, SIBA, SDA, and NASBA. These methods can be coupled to reverse transcription (RT) which enables these methods to amplify RNA targets by first converting the RNA to cDNA through reverse transcription.
CRISPR based diagnostics using Type V (e.g., Cas12) and CasVI (e.g., Cas13) proteins were run using isothermal amplification methods of target nucleic acids to enable sensitive diagnostic assays.
RPA. Recombinase polymerase amplification (RPA) was used to amplify DNA sequences or RNA sequences by including a reverse transcription enzyme in the reaction (RT-RPA). RPA and RT-RPA can be used to generate an amplicon suitable for detection by Type V (e.g. Cas12) Cas proteins.
A “two-pot” DETECTR assay was carried out using RPA and Cas13a by combining the IVT reaction with the RT-RPA or RPA reaction to generate RNA simultaneously with the RPA reaction.
The IVT and Cas13a detection assay reactions were combined with RT-RPA or an RPA reaction to generate and detect RNA simultaneously in a “one-pot” assay.
LAMP. Loop-mediated isothermal amplification (LAMP) was also used for amplifying a DNA sequences or RNA sequences in combination with a reverse transcriptase enzyme (RT-LAMP). LAMP reactions use a combination of four, five, or six primers to amplify the target DNA or cDNA from RNA. During the course of the LAMP reaction, concatemers of amplicons form. If RT-LAMP or LAMP amplicons contain sequence features that support Cas protein recognition (such as PAM or PFS), they can be used as target nucleic acids in CRISPR diagnostics.
Cas12 was also used for the detection of RT-LAMP products.
The primers of an RT-LAMP or LAMP reaction were combined for multiplexed amplification. Because of the formation of concatemers during RT-LAMP and LAMP, it is difficult to differentiate between amplicons in a multiplex RT-LAMP or LAMP reaction by conventional means, as shown in
By including a T7 promoter sequence in the forward inner primers (FIP) or backward inner primers (BIP) of a LAMP or RT-LAMP reaction, the resulting amplicon can be added to an in vitro transcription reaction to generate RNA, as shown in the schematic in
SIBA. Strand invasion based amplification (SIBA) is another isothermal method that can be used.
This example describes optimization of assay conditions for the CRISPR-Cas DETECTR-based diagnostic assays disclosed herein for the detection of target nucleic acids from respiratory viruses (e.g., an influenza virus). The components of the DETECTR reaction, such as protein concentration, crRNA, and buffer components impact the rate and efficiency of the reaction. Optimization of the buffers allows for the development of an assay with increased sensitivity and specificity.
Cas13M26 (LbuCas13a (SEQ ID NO: 131)) performs optimally in DETECTR reactions in buffers with decreased amounts of tRNA without changing the stability of the reaction. Decreasing the amount of tRNA in the reaction or eliminating it completely, increases the efficiency of the Cas13a detection assay without dramatically changing the stability of the reaction in the absence of activator. Buffers in which Cas13a exhibits activity in a DETECTR assay for detection of nucleic acids from a respiratory virus (e.g., influenza virus) lack or have low amounts of urea and SDS. Additionally, Cas13a exhibits activity in DETECTR assays for detection of nucleic acids from a respiratory virus (e.g., influenza virus) in buffers comprising NaCl or KCl, with 30 mM salt or below, and/or with 0-10 mM DTT in buffers containing either NaCl or KCl. Cas13a also exhibits activity, as measured by fluorescence, for a number of reporters, including a “U5” reporter (/5-6FAM/rUrUrUrUrU/3IABkFQ/ (SEQ ID NO: 1)), a “UU” reporter (/56-FAM/TArUrUGC/3IABkFQ/ (SEQ ID NO: 381)), and a reporter with the same nucleotide sequence as the “U5” reporter but with a different fluorophore and quencher, “TYE665U5” (/5-TYE665/rUrUrUrUrU/3IABkRQ/ (SEQ ID NO: 1)). Optimal buffer compositions and pH for Cas13a DETECTR assays include buffers with a pH around 7.5 and buffers imidazole, phosphate, tricine, and SPG.
Cas13a performance is improved in NEBuffer2 (NEBuffer 2.1; 1× Buffer Components, 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 μg/ml BSA, pH 7.9@25° C.) and Cutsmart (1× Buffer Components, 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 100 μg/ml BSA, pH 7.9@25° C.). Cas13a performs optimally in MBufferl. 1× MBuffer1 includes 20 mM imidazole pH 7.5, 50 mM KCl, 5 mM MgCl2, 10 μg/μL BSA, 0.01% Igepal Ca-630, and 5% glycerol. Additionally, Cas13a performance is improved in buffers comprising 5% glycerol, BSA, and NP-40 improve Cas13a DETECTR assay. NP-40 (Igecal-Ca 630) increases the efficiency of the Cas13a detection assay and small amounts of BSA also improve the performance of the assay. Concentrations of 0.05% to 0.0625% NP-40 are the most optimal and concentrations of 2.5 to 0.625 μg/mL BSA are desirable.
Buffers lack compounds that inhibit the performance of the Cas13a DETECTR assay, including: beryllium sulfate, manganese chloride, zinc chloride, tri-sodium citrate, copper chloride, yttrium chloride, 1-6-Diaminohexane, 1-8-diaminooctane, ammonium fluoride, ethanolamine, lithium salicylate, magnesium sulfate, potassium cyanate, and sodium fluoride.
LbCas12a (SEQ ID NO: 27) exhibits optimal activity in DETECTR assays for detection of target nucleic acids from respiratory viruses (e.g., influenza viruses) using buffers with a pH 8.0 and the following buffer types: AMPD, BIS-TRISpropane, DIPSO, HEPES, MOPS, TAPS, TRIS, and tricine buffers. LbCas12a exhibits activity in DETECTR assays with low KCl concentrations (0-40 mM or less than 20 mM salt and less KCl).
A Cas12 variant (SEQ ID NO: 37) performs optimally in a pH of 7.5 and in buffers including DIPSO, HEPES, MOPS, TAPS, imidazole, and tricine. The Cas12 variant performs best at a salt concentration of around 4 mM (ranging from 2-10 nM) and exhibits increased activity in buffers with MgOAc and KOAc (acetate buffers), in comparison to buffers with MgCl and KCl. Additionally, the Cas12 variant is inhibited by heparin and prefers low salt.
Buffers lack specific compounds inhibiting the performance of the Cas12 variant DETECR assay include: benzamidine hydrochloride, beryllium sulfate, manganese chloride, potassium bromide, sodium iodine, zinc chloride, di-ammonium hydrogen phosphate, tri-lithium citrate, tri-sodium citrate, cadmium chloride, copper chloride, yttrium chloride, 1-6 diaminohexane, 1-8-diaminooctane, ammonium fluoride, and ammonium sulfate. Compounds that increase assay performance included: polyvinyl alcohol type II, DTT, DMSO, polyvinylpyrrolidone K15, polyethylene glycol (PEG) 600, and polypropylene glycol 400.
SNP differentiation is stronger for the Cas12 variant along the 3′ end of a crRNA (distal from the PAM). LbCas12a (SEQ ID NO: 27) displays strong mutation sensitivity at all positions along target sequences, and sensitivity on the PAM proximal (complementary to the 5′ end of the crRNA target sequence) end and is more sensitive to mutations in this region and mutation sensitivity is target site dependent.
This example describes a lateral flow test-strip for visual detection of target nucleic acids from a respiratory virus (e.g., an influenza virus) in DETECTR reactions using CRISPR-Cas systems. Visual readouts for the DETECTR reaction are developed to have a low-cost format and be amenable to high-volume manufacturing. Described here are custom-made lateral flow strips. Colloidal gold nanoparticles are conjugated to antibodies and the gold nanoparticles serve as a visual readout in the assay. Two commercially available lateral flow strips are tested including: (1) Millenia Hybridetect 1, TwistDx (UK, now part of Abbott) and (2) PCRD, Abingdon Health (UK).
Results are collected by: (1) visual inspection of the strips, and (2) obtaining a cell-phone-camera picture of the strips. Unlike commercially available lateral flow test strips, the custom-made lateral flow strip design disclosed herein includes a new type of CRISPR-Cas reporter molecule, which is made of (1) a 6-Fluorescein (FAM) moiety; (2) a biotin moiety; and (3) a DNA-based oligo linker, which are irreversibly conjugated to the DETECTR reaction chamber upstream of the reaction.
Lateral Flow Strips for Read Out of a Cas12 variant and LbCas12a. Lateral flow strips are tested for readout of a Cas12 variant (SEQ ID NO: 37) and LbCas12a (SEQ ID NO: 27). Complexing reactions include final concentrations of 40 nM of crRNA per reaction, 40 nM of final protein per reaction, and 500 nM of reporter per reaction. Complexing reactions are incubated at 37 C for 30 min, the reporter substrate is added, and 15 uL of the complexing reactions are aliquoted into PCR tubes. 5 uL of diluted PPR virus PCR product is added and the target (e.g., a sample containing a target nucleic acid from a respiratory virus) and complex are incubated at 37 C for 20 min. 100 uL of Mllenia GenLine Dipstick Assay Buffer (Tween or Triton) is added and the dipstick is inserted into the solution with target and complex. Test strips are photographed and the top band was quantified using ImageJ.
MNT-Lateral Flow, Au NP Conjugation. Anti-FAM and anti-ROX polyclonal antibodies are conjugated to gold nanoparticles for downstream use in the custom made lateral flow strips. Materials include Corning Spin-X UF 500 uL Concentrators and a Gold in a Box Conjugation kit. A 0.5× buffer solution is prepared by diluting PBS, pH 7.2 (1×) in 1:1 with nuclease-free water. 100 ul of the MNT antibody and 100 ul of a FITC antibody are used. Spin concentrators are used to exchange native buffer from 0.1M Tris glycerine, pH 7 with 10% glycerol to 0.5× PBS for both antibodies. Washes with 100 ul of 0.5× PBS are carried out and the concentrators were spun for 1.5 min at 18,000 rcg (×g) for each wash. Antibodies are eluted in 100 ul of 0.5× PBS. Gold conjugation is carried out as per manufacturer's instructions. Tubes are labeled MNT1-10 and FITC1-10 and 7 ul of each antibody was added. Reactions are incubated for 30 min in a shaking incubator at room temperature. The reaction is stopped by adding 50 uL of a BSA blocking buffer to each tubes, and tubes are stored at 4 C.
Lateral Flow Strips for Read Out of LbuCas13a. Lateral flow strips are tested for readout of LbuCas13a. TwistDx lateral flow strips are used to test the FAM-US-Biotin (SEQ ID NO: 1) (rep71 reporter). Assays are run at room temperature at a variety of target concentrations. Complexing reactions include final concentrations of 40 nM of crRNA per reaction, 40 nM of final protein per reaction, and 500 nM of reporter per reaction. Complexing reactions are incubated at 37 C for 30 min. Dilutions of the target are added to the reaction including at 10 nM, 1 nM, 0.1 nM, 0.01 nM, and no target. 30 uL of the complexing reaction is added to the target and incubated for 15 minutes at room temperature. The reaction is placed on ice and 10 uL of the reaction is pipetted directly onto the lateral flow sample area. 50 uL of Milenia GenLine Dipstick Assay buffer is added and the strip was photographed.
Conjugation of 3′Amino Reporter to NHS Beads Using Kit. An NHS FlexiBind Magnetic Bead Kit is used to conjugate the 3′amino modified lateral flow reporter allowing for the intended usage of the lateral flow devices (Milenia Hybrid), where the ligand is detected first and the control line serves as the flow control. The sequence of the reporter used is /56-FAM/*/iBiodT/*AATTAATTAATTAATTAATT/3AmMO/ (SEQ ID NO: 372).
Bead conjugation is carried out as follows. Rep75 is resuspended to 100 μM in Wash/Coupling Buffer (PBS, pH 7.4). 32.5 nmol is delivered from IDT and 5.4 nmol (54 μL) of rep75 is used. 20% NHS FlexiBind Magnetic beads are resuspended by vortexing for 20 seconds. 100 μL of bead slurry is pipetted into a 1.5 mL microcentrifuge tube. Magnetic beads are pelleted on the magnetic stand until the solution became clear. Storage buffer is removed and discarded. 100 μL of ice-cold Equilibration buffer (1 mM HCl) is immediately added. The reaction is removed from the magnet and vortexed for 20 seconds, then placed back on the magnet to pellet beads. The supernatant is removed and discarded and 54 μL of 100 μM rep75 in PBS is added. Beads are incubated at room temperature with interval mixing: 2 min rest, 15 sec mix at 1200 rpm for 2 hours. Tubes are placed in a magnetic stand to allow the beads to migrate to the magnet. Unbound ligand is removed and saved for analysis.
0.5 μL raw reporter is measured in 20 μL NFW vs. 0.5 μL post-conjugation supernatant in 20 μL NFW on a plate reader until it is no longer visibly green. 500 μL of Quench buffer is added, vortexed for 30 seconds, and pelleted with a magnetic rack. The supernatant is discarded and the sample is washed 5 times. Beads are resuspended in 500 μL of Quench Buffer and incubated for 1 hour at room temperature. The beads are pelleted with a magnetic rack and the buffer is removed and discarded. The beads are resuspended in 100 μL of Wash/Coupling Buffer (PBS, pH 7.4) and the beads are kept on ice in dark tube.
Testing uncleaved/unconjugated reporter with lateral flow is carried out using 2× NG-40-B009 Naked Gold Sol beads—40 nm—15 OD—9 mL, FITC antibody (Invitrogen TB265150), anti-IgG (Invitrogen A16098), Streptavadin (NEB N7021S), pH 8.8, and three batches—Batch 1: AU (5 μL)→anti-IgG (μL)→Strep (0.5 μL), Batch 2: AU (5 μL)→strep (1 μL)→anti-IgG (1 μL), and Batch 3: AU (2.5 μL)→strep (1 μL)→anti-IgG (1 μL).
Test beads with a Cas12 variant (SEQ ID NO: 37) by first complexing reaction. Reactions are run with final concentrations of 40 nM crRNA per reaction, 40 nM protein per reaction, and 100, 250, 500, or 1000 nM reporter per reaction. The complex is incubated at 37 C for 30 min. The 40 μM stock of beads is diluted to 1:10 to 4 μM. Reporter beads are added to 5 μL PPRV diluted PCR product or NFW, 15 μL of complexing reaction was added to target. The reaction is incubated at 37 C for 30 min with shaking at 2000 rpm in Thermomixer. Beads are pelleted with magnetic rack for 2 minutes. 10 μL of reaction is transferred to a new tube, 50 μL of Dipstick Assay Buffer is added, and 60 μL diluted reaction is placed on magnet before adding solution to lateral flow strips. Reactions are run on Milenia flow strips.
Gold nanoparticle conjugation to anti-biotin antibody. A 100 ul aliquot of anti-biotin antibody is used, with the antibody suspended in nuclease free water. 7 ul of the dilute antibody in solution is added to tubes and reactions were incubated for 30 min in a shaking incubatory at room temperature. The reaction in each tube is stopped with the addition of 50 ul of the BSA blocked buffer and the tubes were stored at 4 C.
Virus in DETECTR Reactions Using CRISPR-Cas Systems
This example describes a conjugation method for oligonucleotides to peptides/enzymes for downstream use in an invertase coupled assay for amperometric detection of target nucleic acids from a respiratory virus (e.g., influenza virus) in DETECTR reactions using CRISPR-Cas systems. The methods disclosed herein are developed as alternatives to fluorescence and lateral-flow-immunochromatography readouts of DETECTR reactions and include efficient conjugation of an invertase enzyme to a DETECTR reporter using a 3′ thiol modification. The CRISPR-Cas reporter molecule for use in the invertase-coupled assay for amperometric detection of DETECTR reactions includes (1) a 5′-Biotin moiety and (2) a 3′-invertase enzyme. The sequence of the oligo was /5Biosg/TTTTTTTTTTTTTTTTTTTT/3ThioMC3-D/ (SEQ ID NO: 373) and the invertase enzyme is conjugated at the 3′ end. Reagents for the conjugation include invertase from Baker's yeast (S. Cerevisiae), streptavidin magnetic beads, Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-Succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate, SMCC (SMCC), 1M sodium phosphate buffer, pH 7.2, 2-(NMorpholino)ethanesulfonic acid (MES), sodium chloride 5M sterile, and biotin labelled oligos.
Buffer and solution preparation. Buffers include (1) 0.1M Phosphate Buffer, no NaCl, pH 7.2, (2) 0.1M Phosphate Buffer, 0.1M NaCl, pH 7.2, (3) 0.05M MES Buffer, pH 5.5, and (4) 0.05M MES Buffer, with 0.1M NaCl, pH 5.5. TCEP solution, SMCC solution, and invertase solution is prepared from solids. The DNS reagent is also prepared.
Thiol activation of DNA oligo. The thiol-biotin-labelled oligos (15 μL, 1 mM in water) are mixed with TCEP (3 μL, 0.5M in water) in a 1.5-mL microcentrifuge tube. The reaction volume is made up to 30 μL with the addition of 12 μL of eqivalent buffer. The following twelve reactions are prepared: MB406 with low pH, no salt buffer; MB406 with low pH and salt buffer; MB406 with PBS, no salt; MB406 with PBS and salt buffer; MB407 with low pH, no salt buffer; MB407 with low pH and salt buffer; MB407 with PBS, no salt; MB407 with PBS and salt buffer; MB408 with low pH, no salt buffer; MB408 with low pH and salt buffer; MB408 with PBS, no salt; and MB408 with PBS and salt. In each buffer, the volume of DNA oligos is 15 μL, the volume of TCEP was 3 μL, and the volume of buffer was 12 μL. The reaction is incubated for 3-5 hours in the shaking incubator at 37° C. The reaction is stopped by snap freezing in liquid nitrogen. Microcentrifuge tubes are stored at −20° C. until the next step of the reaction. Thiol-activated oligo tubes are removed from the freezer 3 hours prior to conjugation to activated invertase/or other activated proteins/peptides. The tube is first incubated at 37° C. for 3 hours and then used in the conjugation reaction
SMCC activation of invertase enzyme. A fresh solution of invertase lab stock bottle is prepared. 10 mg of solid was weighed in a clean 1.5 mL microcentrifuge tube, 860 μL of buffer A (0.1M NaCl, 0.1M sodium phosphate buffer, pH 7.2) is added to make a solution of 20 mg/mL. 1 mg of SMCC is added to a 1.5 mL microcentrifuge tube and the reaction is initiated by addition of invertase solution (400 μL, 20 mg/mL in 0.1M NaCl, 0.1M sodium phosphate buffer, pH 7.2). The reaction is incubated in the shaking incubator at 37° C. for 24 hours.
Cleanup of SMCC-activated invertase. The reaction is removed from the shaking incubator (37° C.) after 23 hours and 15 minutes. SMCC-activated invertase is washed 8× and resuspended in 400 μL of buffer. Protein is quantified by the BCA method.
Re-activation of thiol-DNA oligo. The oligo is removed from −20° C. and incubated in the shaking incubator at 37° C. The reaction is initiated and incubated in the shaking incubator (37° C.) for 48 hours. The reaction is removed from the incubator and each reaction contained (1) 35 μL of invertase solution, and (2) 30 μL of thiol-DNA oligo solution.
Binding with streptavidin beads. 12.5 μL of streptavidin beads is mixed with 50 μL of the biotinylated DNA oligo previously conjugated with invertase enzyme in a 1.5-mL microcentrifuge tube. The reaction is incubated for 5 minutes at room temperature, beads were washed 5× with 50-μL aliquots of Buffer A on a magnetic rack to remove any unbound DNA oligo from the solution, and eluent from all the washes was checked for invertase activity (and thus inefficient binding between streptavidin and biotin molecules). During the last wash, beads are resuspended with 50 μL of Buffer A and beads were stored at 4° C.
Incubation with DNS/Sucrose. A reaction is prepared containing 5 μL of 20% sucrose, 30 μL DNS reagent, 25 μL of biotynlated DNA with invertase moiety. A color change is observed after incubation at high heat (95 C).
DNA-Invertase Conjugation. Conjugation is carried out using a heterobifunctional linker sulfo-SMCC. To 30 uL of 1 mM thiol-DNA in Millipore water, 2 uL of 1 M sodium phosphate buffer at pH 5.5 and 2 uL of 30 mM TCEP in Millipore water are added and mixed. This mixture is kept at room temperature for 1 hour and then purified by Amicon-10K using Buffer A (0.1 M NaCl, 0.1 M sodium phosphate buffer, pH 7.3, 0.05% Tween-20) without Tween-20 by 8 times. For invertase conjugation, 400 uL of 20 mg/mL invertase in Buffer A without Tween-20 is mixed with 1 mg of sulfo-SMCC. After vortexing for 5 minutes, the solution is placed on a shaker for 1 hour at room temperature. The mixture is then centrifuged and the insoluble excess sulfo-SMCC was removed. The clear solution is then purified by Amicon-100K using Buffer A without Tween-20 by 8 times. The purified solution of sulfo-SMCC-activated invertase is mixed with the above solution of thiol-DNA. The resulting solution is kept at room temperature for 48 hours. To remove un-reacted thiol-DNA, the solution is purified by Amicon-100K 8 times using Buffer A without Tween-20. Conjugation is also carried out using homobifunctional linker PDITC. To 60 uL of 1 mM amine-DNA in Millipore water, 30 uL of Buffer B (0.1 M sodium borate buffer, pH 9.2) are added and mixed. This solution is further mixed with 20 mg of PDITC dissolved in 1 mL DMF. The resulting solution is placed on a shaker and kept at room temperature in the dark for 2 hours. After that, the solution is mixed with 6 mL of Millipore water and 6 mL 1-butanol. After centrifuging for 15 min, the upper organic phase is discarded. The aqueous phase is then extracted with 4 mL 1-butanol three times, and purified by Amicon-10K using Buffer A without Tween-20 for 8 times to produce a PDITC-activated amine-DNA solution. The PDITC activation ratio is over 90% as determined by MALDI-TOF mass spectrometry obtained after desalting the DNA product. Then, 10 mg of invertase is added to the activated DNA solution in Buffer A without Tween-20 to reach a final concentration about 5 mg/mL. The resulting solution is kept at room temperature for 48 hours. To remove un-reacted PDITC-activated amine-DNA, the solution is purified by Amicon-100K 8 times using Buffer A without Tween-20. Tween is not necessary for invertase activity; (2) 1 mg/ml invertase reaction likely finishes after 5 min; (3) 2% sucrose input produces red color at RT after ˜15 min; and (4) DNS is not effective for <0.2% sucrose.
This example describes lateral flow cleavage reporters for CRISPR diagnostics for detection of target nucleic acids from a respiratory virus (e.g., an influenza virus). One design of the Cas reporters disclosed herein involves tethering the Cas reporter to the reaction chamber, upstream of the lateral flow test strip.
Tethered cleavage reporters can also be used to multiplex readouts from CRISPR diagnostics. FAM-biotin and DIG-biotin reporter conjugated to magnetic beads is incubated with a Cas12 variant (SEQ ID NO: 37) for 30 minutes at 37 C in the presence or absence of target DNA (˜0.5 nM) in two separate DETECTR reactions. After the incubation period the magnetic beads are pelleted and the supernatant transferred to a PCRD lateral flow strip (Abingdon Health).
This example describes a lateral flow assay for an influenza CRISPR diagnostic. A DNA or RNA linker is conjugated at the 5′end to biotin-dT/FAM and conjugated at the 3′ end to the substrate of a DETECTR chamber/bead, as shown in
This example describes a lateral flow assay for an influenza CRISPR diagnostic. A DNA or RNA linker is conjugated at the 5′end to biotin-dT/FAM and a second DNA or RNA linker is conjugated at the 5′end to biotin-dT/ROX. Both reporters are conjugated at the 3′ end to the substrate of a DETECTR chamber/bead, as shown in
This example describes diagnosing influenza in a subject with a CRIPSR Cas diagnostic of the present disclosure. A sample is taken from a subject, such as a buccal swab or nasal swab. The subject has an undiagnosed illness. The sample is added to a CRISPR-Cas diagnostic of the present disclosure, for example, the CRISPR-Cas diagnostic of EXAMPLE 17. Guides are designed against influenza virus. The influenza virus is influenza A virus or influenza B virus. The target nucleic acid in the sample, corresponding to influenza, binds to the guide sequence, thus activating transcollateral cleavage of the Cas reporters by a Cas protein. The Cas protein is Cas12, Cas12a, Cas12b, Cas12c, Cas12d, Cas13, Cas13a, or Cas14. The CRISPR diagnostic, thus, reveals a positive result, and the subject is diagnosed with influenza.
This example describes an influenza CRISPR-Cas companion diagnostic of the present disclosure. A sample is taken from a subject, such as a buccal swab or nasal swab. The subject has influenza and has been prescribed and taking a flu therapeutic. The sample is added to a CRISPR-Cas diagnostic of the present disclosure, for example, the CRISPR-Cas diagnostic of EXAMPLE 17. Guides are designed against influenza virus. The influenza virus is influenza A virus or influenza B virus. The target nucleic acid in the sample, corresponding to influenza, binds to the guide sequence, thus activating transcollateral cleavage of the Cas reporters by a Cas protein. The Cas protein is Cas12, Cas12a, Cas12b, Cas12c, Cas12d, Cas13, Cas13a, or Cas14. The CRISPR diagnostic, thus, reveals a result, indicating that the flu therapeutic has not completely eliminated the influenza virus in the subject.
This example shows an invertase-nucleic acid as a detector nucleic for detection of a target nucleic acid from a respiratory virus (e.g., an influenza virus)in a programmable nuclease system.
This example describes assay layouts and workflows for DETECTR reactions for detection of target nucleic acids from a respiratory virus (e.g., an influenza virus). An assay is provided that comprises separate chambers for amplification and reverse transcription versus a programmable nuclease-based detection assay. The programmable nuclease is a Cas12, Cas13, or Cas14. The sample is a biofluid collected by a swab and inserted into a swab collection reservoir. A pump drives the fluidics in the assay moving sample from chamber to chamber. A detectable signal is colorimetric, fluorescence-based, electrochemical and/or generated using an enzyme (e.g., invertase).
This example describes a comparison of the DETECTR assays disclosed herein to the gold standard: PCR-based methods of detecting a target nucleic acid. Samples were either used as a crude prep for DETECTR assays (only lysed) or purified (lysed, bound, washed, and eluted) for PCR-based methods of detection. A DETECTR assay using a programmable nuclease (e.g., a Cas protein) is carried out on the crude sample. The programmable nuclease is activated by the target nucleic acid in a sample to which it binds via a reverse complementary guide RNA. The activated programmable nuclease indiscriminately cleaves a reporter generating a fluorescent detectable signal. Standard PCR-based methods were used to also detect the target nucleic acids in the sample.
This example describes Cas13a detection of target DNA. Cas13a was used to detect a target RT-LAMP DNA amplicon from Influenza A RNA.
Cas13a recognized target ssDNA and target RNA.
Cas13a trans-cleavage activity was found to be specific for RNA reporters when targeting target ssDNA.
Results indicated that Cas13 trans-cleavage was specific for RNA reporters, even when activated by target ssDNA.
Multiple Cas13 family members detected target ssDNA.
Cas13 detection of target ssDNA was robust at multiple pH values.
Cas13 preferences for target ssDNA were found to be distinct from preferences for target RNA.
Cas13a detected target DNA generated by nucleic acid amplification methods (PCR, LAMP).
This example describes assay layouts and workflows for DETECTR reactions. An assay is provided that comprises separate chambers for amplification and reverse transcription versus a programmable nuclease-based detection assay. The programmable nuclease is a Cas12, Cas13, or Cas14. The sample is a biofluid collected by a swab and inserted into a swab collection reservoir. The biofluid sample is tested for the presence of a target nucleic acid from an influenza virus. A pump drives the fluidics in the assay moving sample from chamber to chamber. A detectable signal is colorimetric, fluorescence-based, electrochemical and/or generated using an enzyme (e.g., invertase).
This example describes primer design for combined LAMP and DETECTR reactions for amplification and detection of a target nucleic acid, as provided herein. Strategies for designing primers for use in combined LAMP and DETECTR reactions were tested and evaluated for multiple target nucleic acids. From these experiments, a set of design guidelines was determined to facilitate combined LAMP and DETECTR reactions for DNA nucleic acid targets or RT-LAMP and DETECTR reactions for RNA nucleic acid targets.
Primer sets and guide RNAs for combined LAMP and DETECTR reactions were tested for their sensitivity and specificity to detect the presence of a target nucleic acid in a sample. DETECTR signal, measured as raw fluorescence, was measured for each LAMP primer set with each of three guide RNAs designed for the specific LAMP primer set. DETECTR signal was measured in a sample containing 10000 copies of a target nucleic acid sequence and a sample containing zero copies of a target nucleic acid sequence (negative control) for each LAMP primer and guide RNA pair.
This example describes detection of a target nucleic acid with combined LAMP and DETECTR reactions. Ten LAMP primer sets (#1-#10) for use in RT-LAMP assays were tested for sensitivity and specificity for samples containing a target nucleic acid sequence. Detection following RT-LAMP amplification was performed using either SYTO 9 detection or DETECTR. The sequences of the LAMP primers in each primer set are provided in TABLE 14.
This example describes detection of influenza A and B virus using LAMP and SYTO9. Samples containing either 0, 100, 1000, 10,000, or 100,000 copies of an influenza A virus (IAV) or 0, 100, 1000, 10,000, or 100,000 copies of an influenza B virus (IBV) target nucleic acid sequence were subjected to RT-LAMP amplification using different sets of LAMP primers. Sets of LAMP primers (1, 2, 4, 5, 6, 7, 8, 9, 10, 11, or a negative control) were compared for their ability to specifically amplify the target nucleic acid sequence. Amplification was measured as a time to result using SYTO9. A decreased time to result is indicative of a sample positive for the target nucleic acid sequence.
Each reaction RT-LAMP reaction was performed in the presence of 1× NEB IsoAmp Buffer, 4.5 mM MgSO4, 6.4 U/μL Bst 2.0 (NEB), 0.75 μL Warmstart RTx reverse transcriptase, 1 μL 10×0 primer mix, and 0.2 μL SYTO9 per 10 μL reaction in nuclease free water.
This example describes detection of influenza A virus using LAMP and DETECTR. Samples containing an influenza A virus (IAV) target nucleic acid sequence or lacking the IAV target nucleic acid sequence were subjected to RT-LAMP amplification using different sets of LAMP primers. Sets of LAMP primers were compared for their ability to specifically amplify the target nucleic acid sequence. Presence or absence of the target nucleic acid in the sample was subsequently measured using DETECTR. DETECTR signal, measured by an increase in fluorescent signal upon activation of a programmable nuclease, was observed over time. An increase in fluorescence indicates the presence of the target nucleic acid sequence.
Each RT-LAMP reaction was performed in the presence of 1× NEB IsoAmp Buffer, 4.5 mM MgSO4, 1.4 mM dNTPs (NEB), 6.4 U/μL Bst 2.0 (NEB), 1.5 μL Warmstart RTx, and 2 μL 10× primer mix per 20 μL reaction in nuclease-free water. Each DETECTR reaction was performed in the presence of lx Processing Buffer, 250 nM crRNA, and 200 nM Sr-WT LbCas12a (SEQ ID NO: 27) programmable nuclease in nuclease-free water.
This example describes detection of a SNP using LAMP and DETECTR. Strategies for designing primers for use in combined LAMP and DETECTR reactions to detect SNPs were tested and evaluated for multiple target SNPs. From these experiments, a set of design guidelines was determined to facilitate combined LAMP and DETECTR reactions for DNA nucleic acid targets or RT-LAMP and DETECTR reactions for RNA nucleic acid targets.
The target sequence was detected using a guide RNA (crRNA only) to detect either the A allele with the first PAM site (SNP Position 9, “A SNP”), the G allele with the first PAM site (SNP Position 9, “G SNP”), the A allele with the second PAM site (SNP Position 14, “A SNP”)or the G allele with the second PAM site (SNP Position 14, “G SNP”). Four guide RNAs designed for each condition were used. The guide RNAs used for the detection of the two SNP alleles relative to the two PAM sites are presented in TABLE 19. The guide RNA corresponding to SEQ ID NO: 255 was designed to detect the A allele at position 9, the guide RNA corresponding to SEQ ID NO: 256 was designed to detect the G allele at position 9, the guide RNA corresponding to SEQ ID NO: 257 was designed to detect the A allele at position 14, and the guide RNA corresponding to SEQ ID NO: 258 was designed to detect the G allele at position 14. A high fluorescence signal was detected for the G allele in the presence of the position 9 G SNP guide RNA (SEQ ID NO: 256, top left) and the A allele in the presence of the position 9 A SNP guide RNA (SEQ ID NO: 255, bottom right). Minimal fluorescence signal was detected for the G allele in the presence of the position 9 A SNP guide RNA (SEQ ID NO: 255, top right) and the position 9 A allele in the presence of the G SNP guide RNA (SEQ ID NO: 256, bottom left). This indicates that the position 9 G SNP and position 9 A SNP guide RNAs show specificity for the G allele and A allele, respectively. The position 14 A SNP guide RNA (SEQ ID NO: 257) and the position 14 G SNP guide RNA (SEQ ID NO: 258) detected both alleles, as shown by high fluorescence signal when detecting the SNP with the position 14 A SNP or G SNP guide RNAs, independent of the target sequence present.
This example describes RT-LAMP DETECTR reactions for the detection of coronavirus. SARS-CoV-2 target sequences were designed using all available genomes available from GISAID. Briefly, viral genomes were aligned using Clustal Omega. Next, LbCas12a target sites on the SARS-CoV-2 genome were filtered against SARS-CoV, two bat-SARS-like-CoV genomes and common human coronavirus genomes. Compatible target sites were finally compared to those used in current protocols from the CDC and WHO. LAMP primers for SARS-CoV-2 were designed against regions of the N-gene and E-gene using PrimerExplorer v5 (https://primerexplorer.jp/e/).
Target RNAs were generated from synthetic gene fragments of the viral genes of interest. First a PCR step was performed on the synthetic gene fragment with a forward primer that contained a T7 promoter. Next, the PCR product was used as the template for an in-vitro transcription (IVT) reaction at 37° C. for 2 hours. The IVT reaction was then treated with TURBO DNase (Thermo) for 30 minutes at 37° C., followed by a heat-denaturation step at 75° C. for 15 minutes. RNA was purified using RNA Clean and Concentrator 5 columns (Zymo Research). RNA was quantified by Nanodrop and Qubit and diluted in nuclease-free water to working concentrations.
DETECTR assays were performed using RT-LAMP for pre-amplification of viral or control RNA targets and LbCas12a for the trans-cleavage assay. RT-LAMP was prepared with a MgSO4 concentration of 6.5 mM and a final volume of 10 μL. LAMP primers were added at a final concentration of 0.2 μM for F3 and B3, 1.6 μM for FIP and BIP, and 0.8 μM for LF and LB. Reactions were performed independently for N-gene, E-gene, and RNase P using 2 μL of input RNA at 62° C. for 20 minutes.
For LbCas12a (SEQ ID NO: 27) trans-cleavage, 50 nM LbCas12a (available from NEB) was pre-incubated with 62.5 nM gRNA in 1X NEBuffer 2.1 for 30 minutes at 37° C. After formation of the RNA-protein complex, the lateral flow cleavage reporter (/56-FAM/TTATTATT/3Bio/ (SEQ ID NO: 9), IDT) was added to the reaction at a final concentration of 500 nM. RNA-protein complexes were used immediately or stored at 4° C. for up to 24 hours before use.
After completion of the pre-amplification step, 2 μL of amplicon was combined with 18 μL of LbCas12a-gRNA complex and 80 μL of 1× NEBuffer 2.1. The 100 μL LbCas12a trans-cleavage assay was allowed to proceed for 10 minutes at 37° C.
A lateral flow strip (Milenia HybriDetect 1, TwistDx) was then added to the reaction tube and a result was visualized after approximately 2-3 minutes. A single band, close to the sample application pad indicated a negative result, whereas a single band close to the top of the strip or two bands indicated a positive result.
The patient optimized DETECTR assays were performed using RT-LAMP method as described above with the following modifications: A DNA binding dye, SYTO9 (Thermo Fisher Scientific), was included in the reaction to monitor the amplification reaction and the incubation time was extended to 30 minutes to capture data from lower titre samples.
The fluorescence based patient optimized LbCas12a trans-cleavage assays were performed as described above with modifications; 40 nM LbCas12a was pre-incubated with 40 nM gRNA, after which 100 nM of a fluorescent reporter molecule compatible with detection in the presence of the SYTO9 dye (/5Alex594N/TTATTATT/3IAbRQSp/ (SEQ ID NO: 9)) was added to the complex. 2 μL of amplicon was combined with 18 μL of LbCas12a-gRNA complex in a black 384-well assay plate and monitored for fluorescence using a Tecan plate reader.
This example describes the screening of primer sets for amplification of a SARS-CoV-2 target site. A region of the coronavirus RNA genome corresponding to the viral N-gene was amplified using different LAMP primer sets (sett through set 11). Samples containing either 1.5 pM, 5 fM, or 0 fM SARS-CoV-2 RNA were amplified with each primer set. SARS-CoV-2 RNA in each sample was reverse transcribed using a warmstart reverse transcriptase (“Warmstart RTx”) and LAMP amplified using a Bst 2.0 DNA polymerase. The assay was performed at 60 degrees C. for 60 minutes.
A DETECTR assay was performed on each amplified sample, and the time to result was determined. Sequences were detected using a gRNA sequence corresponding to R1763 directed to the N-gene of SARS-CoV-2 and a Cas12 programmable nuclease corresponding to LbCas12a. The DETECTR assay was sensitive for the amplified SARS-CoV-2 target sequence for all tested primer sets. Sequences of the gRNAs used in this example are provided in TABLE 20.
In a second assay, primer sets directed to the E-gene of Sarbeco (detected with gRNAs R1764 and R1765) and the N-gene of Sarbeco (detected with R1767).
A control primer set for amplifying RNase P was also tested.
This example describes the specificity of detection of a SARS-CoV-2 target nucleic acid. A sample containing target RNA corresponding to SARS-CoV-2 was amplified as using primer set 1 as described in EXAMPLE 2. gRNAs were screened for compatibility with different primer sets designed to amplify either the N-gene or the E-gene of SARS-CoV-2.
Samples containing either 5 fM or 0 fM SARS-CoV-2 RNA were detected using a DETECTR assay. Samples were detected using LbCas12a and either a gRNA R1763 directed to the N-gene of SARS-CoV-2 or a gRNA R1766 directed to the N-gene of SARS-CoV. Sequences of the gRNAs used in this example are provided in TABLE 20.
This example describes the limit of detection of SARS-CoV-2. Samples containing decreasing copy numbers of SARS-CoV-2 target nucleic acid were detected using a DETECTR reaction.
This example describes multiplexing SARS-CoV-2 primer sets for detection of SARS-CoV-2. Samples containing target nucleic acids were amplified using a combination of primer sets directed to one or more of SARS-CoV-2 or RNase P. Primer sets directed to SARS-CoV-2 are denoted by “set” with a number.
This example describes the sensitivity of a DETECTR assay to distinguish three coronaviruses. Samples containing 250 pM of either RNA corresponding to the N-gene of SARS-CoV-2, the N-gene of SARS-CoV, or the N-gene of bat-SL-CoV45. Samples were amplified at detected as described in EXAMPLE 2. Samples were detected using each of a gRNA directed to the N-gene of SARS-CoV-2 (“R1763”), a gRNA directed to the N-gene of SARS-CoV (“R1766”), or a gRNA directed to the N-gene of a Sarbeco coronavirus (“R1767”). Sequences of the gRNAs used in this example are provided in TABLE 20.
This example describes the sensitivity of detection of the E-gene of three coronaviruses. Samples containing 250 pM of either RNA corresponding to the E-gene of SARS-CoV-2, the E-gene of SARS-CoV, the E-gene of bat-SL-CoV45, or the E-gene of bat-SL-CoV21. Samples were amplified at detected as described in EXAMPLE 2. Samples were detected using each of a first gRNA directed to the E-gene (R1764), or a second gRNA directed to the E-gene (R1765). Sequences of the gRNAs used in this example are provided in TABLE 20.
This example describes the detection of a coronavirus using a lateral flow DETECTR reaction.
A sample comprising an RNA target sequence from a coronavirus was amplified using isothermal amplification. Samples containing either 0 fM (“−”) or 5 fM (“+”) of in vitro transcribed coronavirus N-gene were amplified for 60 minutes using a reverse-transcription LAMP (RT-LAMP) amplification assay. A DETECTR reaction was performed using a Cas12 variant (SEQ ID NO: 37) for either 0 min, 2.5 min, 5 min, or 10 min.
This example describes the detection of SARS-CoV-2 using a lateral flow DETECTR reaction.
This example describes the testing of clinical samples for SARS-CoV-2 using a DETECTR reaction. Clinical samples were amplified using RT-PCR and detected using LbCas12a. Samples were detected using gRNA (“crRNA”) directed to either the N-gene or the E-gene of SARS-CoV-2 or RNase P (negative control).
Clinical samples of patients either positive or negative for SARS-CoV-2 were assayed using a lateral flow DETECTR reaction. Samples were amplified and reverse transcribed using RT-PCR and detected using a Cas12 programmable nuclease. A negative control sample (“NTC”) was also assayed. The DETECTR reaction was performed for 5 min.
This example describes buffer screening for improved RT-LAMP amplification and detection. Samples containing either HeLa total RNA (“total RNA”), SARS-CoV-2 N-gene RNA and HeLa total RNA (“N-gene +total RNA”) or no target (“NTC”) were amplified using RT-LAMP under different buffer conditions.
This example describes the limit of detection of SARS-CoV-2 in a DETECTR assay. DETECTR reactions were performed with different copy numbers of SARS-CoV-2 viral genomes.
This example describes the target specificity of a multiplexed RT-LAMP amplification with DETECTR reaction.
This example describes coronavirus strain specificity of N-gene and E-gene gRNAs. Guide RNAs were designed to specifically detect the N-gene of SARS-CoV-2. Guide RNAs were also designed to detect the E-gene in three SARS-like coronavirus strains (SARS-CoV, bat SARS-like coronavirus (bat-SL-CoVZC45), and SARS-CoV-2). Synthetic in vitro transcribed (IVT) SARS-CoV-2 RNA gene targets were spiked into nuclease-free water. Samples were detected with a CRISPR-Cas12 based detection assay using LbCas12a (SEQ ID NO: 27). DETECTR assays included an RT-LAMP reaction at 62° C. for 20 min and Cas12 detection reaction at 37° C. for 10 min. Primers for target generation, qPCR, and LAMP amplification are provided in TABLE 23.
Guide RNAs were able to distinguish SARS-CoV-2 without cross-reactivity with related coronavirus strains using the N gene gRNA and with the expected cross-reactivity for the E gene gRNA.
This example describes specific and broad detection of coronaviruses using a lateral flow DETECTR assay. Lateral flow DETECTR assays can be performed with minimal equipment within appropriate biosafety laboratory requirements.
The DETECTR assay can be run within 30 to 40 minutes and visualized on a lateral flow strip. Conventional RNA extraction or sample matrix can be used as an input to DETECTR (LAMP pre-amplification and Cas12-based detection for N gene, E gene and RNase P), which is visualized by a fluorescent reader or lateral flow strip. The SARS-CoV-2 DETECTR assay was considered positive if there was detection of both the E and N genes, or presumptive positive if there was detection of either the E or N gene. This interpretation is consistent with that of current FDA Emergency Use Authorization (EUA) guidance and recently approved point-of-care diagnostics under the EUA.
This example describes amplification and detection of patient samples directly from raw sample matrix. The capability of the RT-LAMP assay to amplify SARS-CoV-2 nucleic acid directly from raw sample matrix was assessed. Samples consisting of nasal swabs from asymptomatic donors placed in universal transport medium (UTM) or phosphate buffered saline (PBS) and spiked with SARS-CoV-2 IVT target RNA were assayed using RT-LAMP DETECTR reactions. Since nasal swabs are more frequently collected in universal transport medium (UTM) than in phosphate buffered saline (PBS), the effect of running the assay from nasal swab sample matrix consisting of UTM buffer was evaluated. Nasal swabs from asymptomatic donors were collected in UTM or PBS.
This example describes the limit of detection of a DETECTR assay for SARS-CoV-2. Using IVT SARS-CoV-2 target RNA spiked into donor nasal swab sample matrix in PBS, the analytic limits of detection (LoD) of the DETECTR assay was compared relative to the US FDA Emergency Use Authorization (EUA)-approved CDC assay (running tests for 2 of the 3 targets, N2 and N3) for detection of SARS-CoV-2. Five 10-fold serial dilutions of in vitro transcribed viral RNA were spiked into sample matrix at concentrations ranging from 101-105 copies/mL, with 6 replicates at each dilution for the DETECTR assay, and 3 replicates at each dilution for the CDC assay.
The analytic limit of detection of the RT-LAMP DETECTR reaction was compared relative to the qRT-PCR detection assay used by the US FDA Emergency Use Authorization-approved CDC assay for detection of SARS-CoV-2. A standard curve for quantitation was constructed using 7 dilutions of a control IVT viral nucleoprotein RNA (“CDC VTC nCoV Transcript”), with 3 replicates at each dilution, and detected using the CDC protocol (
The limit of detection (LoD) was measured for detection of SARS-CoV-2 using a lateral flow device.
This example describes the effects of incubation time in a DETECTR assay for SARS-CoV-2. Samples were amplified using RT-LAMP and detected using LbCas12a (SEQ ID NO: 27). The effect of the Cas12 reaction incubation time on signal was tested.
This example describes detection of SARS-CoV-2 in patient samples using a DETECTR assay. Extracted RNA from nasal swab samples collected from six patients with documented SARS-CoV-2 infection, nasal swab samples from 15 patients with other influenza or coronavirus infections, and nasal swab samples from five healthy donors were tested. RNA extracts from patients with influenza (n=4) and other human coronavirus infections (common human seasonal coronavirus infections (OC34, HKU1, 229E and NL63, n=7)) were compared to in vitro transcribed SARS-CoV-2 target RNA spiked into nasal swab matrix in UTM and RNA extracted from nasal swabs from 2 SARS-CoV-2 infected patients. Samples were detected using SARS-CoV-2 DETECTR assay with fluorescence and lateral flow strip readouts
SARS-CoV-2 was detected in 9 of the 11 patient swabs and did not cross react with the other respiratory viruses. The two negative swabs from COVID-19 patients were confirmed to be below the established limit of detection.
Given the 100% concordance between lateral flow and fluorescence-based readouts shown in
Relative to the CDC qRT-PCR protocol, the SARS-CoV-2 DETECTR assay was 90% sensitive and 100% specific for detection of the coronavirus in nasal swab samples, corresponding to positive and negative predictive values of 100% and 91.7%, respectively.
SARS-CoV-2 DETECTR assay (RT-LAMP+Cas12a) was evaluated on IVT RNA products from SARS-CoV-2, SARS-CoV, bast-SL-CoVZC45, and clinical samples from common human coronaviruses.
This example describes improved detection of an RNase P POP7 control gene with modified LAMP primers and gRNA. Samples containing RNase P POP7 RNA were assayed using RT-LAMP and DETECTR reactions to assess the amplification and detection efficiency of primer sets and gRNAs directed to RNase P POP7. Samples containing either 0.16 ng/μL total RNA or 0 ng/μL total RNA were amplified by RT-LAMP with different primer sets at 60° C. for 60 minutes.
A DETECTR reaction was performed on the amplicons generated by RT-LAMP. Samples were detected using gRNAs corresponding to R779 (SEQ ID NO: 330), R780 (SEQ ID NO: 332), or R1965 (SEQ ID NO: 331).
The limit of detection was then tested for RNase P POP7 amplified using RT-LAMP with primer set 1 (SEQ ID NO: 360-SEQ ID NO: 365) or primer set 9 (SEQ ID NO: 366-SEQ ID NO: 371) and detected with R779 gRNA (SEQ ID NO: 330) or R1965 gRNA (SEQ ID NO: 331).
This example describes a viral lysis buffer for lysis and amplification of a coronavirus. Nasal swab or saliva samples are collected from individuals suspected of having a coronavirus infection. Nasal swab and saliva samples are suspended in a viral lysis buffer formulated to lyse the viral capsids and release the viral genome. The viral lysis buffer is compatible with RT-LAMP amplification of the viral genome and DETECTR detection of a target nucleic acid, providing a one-step sample preparation solution for a coronavirus DETECTR reaction.
This example describes detection of a SNP using a DETECTR assay on a microfluidic cartridge. This assay was performed on a microfluidic cartridge shown in
The first heater of the manifold was set to 60° C., and the second heater was set to 37° C. The sample was incubated for 30 minutes at 60° C. After 30 minutes, a first pump in the manifold was initiated to pump the LAMP buffer with the sample through the cartridge. A second pump in the manifold was initiated to push 95 μL of the DETECTR solution into the detection chamber. The sample was incubated at 37° C. for 30 minutes. Fluorescence was visualized using a black box fluorescence detector.
A control assay was performed in microcentrifuge tubes using a heating block. In a first tube, 5 μL of a sample from a blue-eyed individual was combined with 45 μL of a LAMP master mix solution. In a second tube, 5 μL of a sample from a brown-eyed individual was combined with 45 μL of a LAMP master mix solution. Samples were incubated for 30 minutes at 60° C. in a mini dry bath. 5 μL of each amplified sample was transferred to 95 μL of a 1× RNP solution for detection of A and G SNPs. The reactions were transferred to a 37° C. heat block.
This example describes amplification and detection of a SNP in a microfluidic cartridge. These assays were performed in the microfluidic cartridge illustrated in
PMMA layers of the cartridge were cleaned by immersion in RNAse Zap for 20 minutes and washing of remnants of the cleaning solution by washing twice in nuclease free water. The cartridge was dried using a stream of nitrogen. The layers of the cartridge were assembled. The top half of the CRISPR reaction workflow was blocked with high sol epoxy and dried for 20 minutes until clear. 80 μL of LAMP master mix was pre-mixed in a microcentrifuge tube with 10 μL of primer mix and 10 μL of pure DNA extract. The solution was mixed by pipetting up and down. 70 μL of this solution was loaded into the amplification chamber of the cartridge using a pipette. The chamber was sealed using a small rectangular piece of PCR adhesive (Biorad, MSB-1001).
The cartridge was placed into a heating manifold, and the aluminum block was heated to an on-chip temperature of 60° C. The sample was incubated at 60° C. for 30 minutes to amplify the sample using LAMP. 100 μL of CRISPR reagent containing a blue-eye gRNA was added to the lower DETECTR chamber. The top and bottom chambers were sealed with small rectangular pieces of PCR adhesive. The CRISPR reagents were mixed with 5 μL of the amplified sample by actuating a valve in the cartridge. The manifold was covered with a shroud of 3D printed APS to block light. The aluminum block was heated to an on-chip temperature of 37° C. The CRISPR reaction was incubated for 30 minutes at 37° C. The resulting fluorescence was observed by eye.
The assay was repeated as described above using the cartridge illustrated in
This example describes amplification and detection of a SNP in a revised microfluidic cartridge. This assay was performed on a microfluidic cartridge illustrated in
40 μL of LAMP master mix was pre-mixed in a microcentrifuge tube with 5 μL of primer mix and 5 μL of pure DNA extract. The solution was mixed by pipetting up and down. 50 μL of this solution was loaded into the amplification chamber of the cartridge using a pipette. The chamber was sealed using a small rectangular piece of PCR adhesive (Biorad, MSB-1001). 95 μL of the CRISPR reagent solution containing a Cas12 variant (SEQ ID NO: 37) and a gRNA directed to a brown-eye SNP was added to the lower DETECTR chamber, and 95 μL of a negative reagent solution (5× MBuffer3) was added to the upper DETECTR chamber. The chamber was sealed using a small rectangular piece of PCR adhesive (Biorad, MSB-1001).
The cartridge was assembled on a heating manifold, and the aluminum block was heated to an on-chip temperature of 60° C. at the amplification chamber. Heating was initiated 2 minutes prior to beginning the assay. Amplification was performed at 60° C. for 30 minutes. The valve of the cartridge was actuated to mix the CRISPR reagent with 5 μL of the amplified sample. The manifold heater of the detection chamber was heated to 37° C. without pre-heating. The DETECTR reaction was performed at 37° C. for 30 minutes, and the resulting fluorescence was observed by eye. The chambers were imaged by illuminating with either an LED from a mini PCR kit or an LED from ThorLabs.
The assay was repeated on a new cartridge of the same design with the following modifications: the CRISPR reagents were not preloaded into the device, because the heater was still warm from the previous run, and the amplification and detection steps were run for 15 minutes instead of 30 minutes.
A third assay was performed on a microfluidic cartridge illustrated in
This example describes use of a microfluidic device for a DETECTR reaction. A microfluidic cartridge as illustrated in any of
At the end of the 30-minute 60° C. LAMP incubation, the solenoid valve opens and the peristaltic pump #1 engages at 100% PWM for 10 seconds. The LAMP buffer is pumped through the valve to the intersection of the serpentine channels leading to the DETECTR reaction chambers and the straight channels leading to the DETECTR reagent reservoirs. The serpentine channel leading to the DETECTR reaction chambers has a larger cross-sectional area than the channel leading to the DETECTR reagent reservoirs. This is intended to reduce the fluidic resistance in the serpentine channels and direct all of the buffer towards the DETECTR reaction chambers. However, throughout this study (testing 23+ chips), the buffer has split both ways nearly every time, with approximately half the buffer volume going the wrong way. In the next fluidic step, the solenoid valve closes and DETECTR reagent is pumped towards the DETECTR reaction chambers, collecting the LAMP product along the way. This provides some mixing as both buffers travel through the serpentine channels simultaneously, but this process also creates bubbles that can get carried to the DETECTR chamber.
To prevent bubbles from interfering with fluorescence measurements during DETECTR, a larger volume of buffer is loaded into the reservoirs than the reaction chambers can fit and use a longer pumping time than necessary. This ensures that the chambers are completely filled with reagent and all bubbles have been popped. The DETECTR reaction chambers have a 70 μL volume, and 25 μL LAMP plus 95 μL DETECTR reagent are delivered into each chamber. The second fluidic step (DETECTR reagent to the DETECTR reaction chambers) takes about 20-30 seconds to deliver all the buffer, but this step is run for 45 seconds. This results in completely full DETECTR reaction chambers, with the excess reagents backed up in the serpentine channels. In addition to bubbles, if the DETECTR reaction chambers are not completely filled, condensation forms on the top of the chamber during the 37° C. incubation, which also interferes with fluorescence measurements taken from above.
This example describes thermal testing of a microfluidic device for a DETECTR reaction. The thermal performance of a heating manifold was tested by measuring the time to temperature and the accuracy of heating to the setpoints with thermocouples submerged within the buffer. Under standard assay temperature setpoints (60° C. LAMP/37° C. DETECTR), the LAMP buffer heats to 60° C. in 8.5 minutes, but the DETECTR buffer reaches a maximum temperature of 34° C. at around 21 minutes. This is somewhat counterintuitive, since it takes longer to hit a lower temperature (and the DETECTR buffer does not reach the setpoint temperature). To hit a specific temperature, the heater controller varies the amount of time it spends in the on state. This state switching is quantified by the pulsed width modulation (PWM) value, the percentage of a given unit of time it spends in the on state. The heater controller also samples the temperature of the heater for feedback on the difference between the current temperature and the setpoint temperature. The larger the difference between those two values, the higher the resulting PWM value will be. As the heater temperature approaches the setpoint, the PWM value drops to slow the rate of change and avoid overshooting the setpoint temperature. The difference between the room temperature heater and the LAMP setpoint is about 35° C., while the difference between the DETECTR heater and its setpoint is about 12° C. The LAMP incubation heats with maximum PWM values around 20%, and the DETECTR incubation heats with maximum PWM values around 12%. Our current setup is designed with a larger emphasis on accuracy and not overshooting the setpoint temperature than heating the buffer to assay temperature quickly.
Specific PWM values can be used to heat to our setpoint temperatures faster. However, this is a manual process and can result in overshooting the target temperatures and damaging the manifold prototype and melting the microfluidic chip. With the LAMP heater PWM value set to 100%, the LAMP buffer (measured by thermocouple) heats to 60° C. in 90 seconds, but the heater temperature hits 100° C. With the DETECTR heater PWM set to 100%, the DETECTR buffer heats to 37° C. in 60 seconds, and the heater hits 80° C. Turning the heater off when the DETECTR buffer hits 37° C. results in a maximum buffer temperature of around 60° C. the temperature of the DETECTR side of the chip rises during the 30-minute 60° C. LAMP incubation so that it is higher than room temperature. It varies from time to time, but it is usually between 25-29° C. by the beginning of the DETECTR side.
This example describes detection of a HERC2 SNP using a microfluidic cartridge. A primer mix containing 2 μM F3 primer, 2 μM B3 primer, 16 μM FIP primer, 16 μM BIP primer, 8 μM LF primer, and 8 μM LB primer in nuclease free water was prepared. A complexing reaction containing 1× MBuffer3, 40 nM crRNA, and 50 nM Cas12 variant (SEQ ID NO: 37) was prepared. 40 nM reporter substrate was added after incubating at 37° C. for 30 minutes. A LAMP mix containing 1× IsoAmp Buffer, 4.5 mM MgSO4, dNTPs, and 1× primer mix was prepared. DETECTR reagents were loaded into a microfluidic cartridge and wells were sealed with PCR tape. LAMP mix was mixed with primers and loaded into the cartridge. The narrow end of the Chip Shop tank was covered with parafilm and inserted into the luer connection above the LAMP reaction chamber. The Chip Shop tank was loaded with 200 μL of 20 mM NaOH. The cartridge was inserted into the heating manifold and screws were tightened. A buccal swab was added to the tank, gently agitated, and incubated for 2 minutes. A Drummond micropipette was used to deliver 10 μL of lysed sample through parafilm into LAMP reaction chamber. The tank was removed and the chamber was sealed with qPCR tape cut to size.
Another assay was performed. Solutions were prepared as described above, and samples were run on a microfluidic cartridge shown in
This example describes detection of a coronavirus using a microfluidic cartridge. A complexing reaction containing 1× MBuffer3, 40 nM crRNA, and 50 nM Cas12 variant (SEQ ID NO: 37) was prepared. 40 nM reporter substrate was added after incubating at 37° C. for 30 minutes. 95 μL DETECTR reagents were loaded into each DETECTR reagent well and sealed with qPCR tape. A tube of N Gene LAMP master mix (537 μL) was mixed with 32 μL of 100 mM MgSO4 and 40 μL of mixture was loaded into a cartridge. 10 μL of Twist SARS-Cov-2 standard was added at various copies/μL or 1× TE as a negative control to LAMP reaction chamber. The cartridge was inserted into the manifold and tightened. The LAMP reaction chamber was sealed with qPCR tape. Temperatures were set (62° C. LAMP, 40° C. DETECTR (to account for thermal offset)) and automated workflow was initiated. A 3D-printed optical cover was placed on the cartridge to minimize optical noise. DETECTR measurements were taken at 0 min, 2 min, 5 min, 10 min, 20 min, and 30 min. The copy number of RNA in the LAMP reaction was varied in order to estimate the lower limit of detection in the device.
The assay was repeated.
This example describes the turnaround time of an influenza B DETECTR assay in a microfluidic cartridge. A primer mix containing 2 μM F3 primer, 2 μM B3 primer, 16 μM FIP primer, 16 μM BIP primer, 8 μM LF primer, and 8 μM LB primer in nuclease free water was prepared. A complexing reaction containing 1× MBuffer3, 40 nM crRNA, and 50 nM Cas12 variant (SEQ ID NO: 37) was prepared. 40 nM reporter substrate was added after incubating at 37° C. for 30 minutes. 95 μL DETECTR reagents were loaded into each DETECTR reagent well and sealed with qPCR tape. 40 μL of LAMP mixture was added to the cartridge. 2 μL of 1 pM IBV target was added to 198 μL of viral lysis buffer and loaded into a Chip Shop tank. A Drummond micropipette was used to deliver 10 μL of lysed sample through parafilm into LAMP reaction chamber. The tank was removed and the chamber was sealed.
This example describes the use of glass capillaries in a DETECTR reaction. Glass capillaries can enable fluid flow by passive capillary action, thereby obviating the need for power-driven flow (e.g., with mechanical pumps). Glass capillaries are also capable of long-term, stable reagent storage.
The reagents required for a DETECTR reaction are provided in inside of the capillaries in dry form. Hydrating the capillaries solubilizes the reagents, and allows them to be eluted from the capillaries into a collector compartment or container. CRISPR-Cas complexes may be stored and recollected in this fashion without loss of activity.
DETECTR reagent mixes were prepared by pre-complexing a guide nucleic acid of SEQ ID NO: 374 with 5 μM of a programmable nuclease with SEQ ID NO: 37 in 5× MBuffer2 (20 mM Tris HCl, pH8, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol, 50 ug/mL Heparin). 23.5 mm capillaries with 20 μl volume capacities were loaded with 0.5 μl droplets of a reagent mix and then dried at room temperature overnight. After 212 days, the capillaries were rehydrated with 20 μl aliquots of 5× MBuffer2 containing either 0 μM or 0.170 μM ssDNA substrates with fluorescent reporters and either 0 μM, 0.1 μM, or 1 μM target nucleic acid. The sequences of the guide nucleic acid, ssDNA substrates, and target nucleic acid are provided in TABLE 24 below. Following 2 minutes of room temperature rehydration, the contents of each capillary were expunged into separate wells on a 384-well plate. The wells were incubated at 37° C. for 90 minutes, during which time a fluorescence readout was monitored from each well.
The results of the DETECTR experiment are shown in
This example describes a device that is designed to mix reagents for a nucleic acid amplification reaction and a CRISPR reaction. Amplification reactions and CRISPR reactions often require separate buffers and conditions. Thus, performing sequential amplification and CRISPR reactions on a single sample can require exposing the sample to the surrounding environment. This example describes a multicompartment spin-column that can move a sample through separate compartments containing different reagents while remaining sealed to reduce contamination.
The spin-column can have the structure illustrated in
The spin-column can be used in the method illustrated in
This example describes reporter molecules with electrochemically detectable moieties for use in DETECTR reactions. The reporter molecules are ssDNA containing modified thymine nucleobases conjugated to ferrocene moieties and a fluorescent moiety (e.g., fluorescein) conjugated to the 5′ end via a phosphodiester linkage. Reporter molecules may be biotinylated at the 3′ end. The sequences of two ssDNA reporter molecules are 5′-YXXTTATTXX-3′ (SEQ ID NO: 391) and 5′-YXXTTATTATTXXZ-3′ (SEQ ID NO: 392), wherein X is ferrocene labeled thymidine (
In a DETECTR reaction, the reporter molecules may undergo transcollateral cleavage from a programmable nuclease (e.g., a Cas12 variant having a sequence of SEQ ID NO: 37). Reporter molecule cleavage mobilizes electrochemically detectable, ferrocene containing ssDNA subunits. Ferrocene has a relatively high oxidation potential, and thus can be potentiometrically detected against a background of low oxidation potential biomolecules. The magnitude of the electrochemical signal increases with cleavage of the reporter molecules. In contrast, the intensity of the fluorescence signal from the reporter molecules is invariant to the degree of transcollateral cleavage. Thus, a fluorescence readout can be used to calibrate the electrochemical measurements by quantifying the total concentration of reporter molecules present, and the combination of electrochemical and fluorescence measurements can be used to determine the fraction of reporter molecules which have been cleaved. The biotin serves as a capture moiety for the reporter or fragments of the reporter (e.g., with streptavidin).
The assays were performed with a 5′-YXXTTATTXX-3′ (SEQ ID NO: 391) reporter oligonucleotide, a programmable nuclease targeting HERC2, and a HERC2 target nucleic acid. Detection was performed with a DropSens p.STAT ECL instrument and DropSens screen-printed carbon electrodes. Aliquots of the DETECTR reaction were collected at multiple time points after its initiation.
This example describes a device capable of performing multiple amplification and CRISPR reactions on a sample. The device is capable of dividing a sample to perform multiple, distinct sequences of amplification and CRISPR reactions on different aliquots of a single input sample. The device houses a microfluidic chip containing multiple compartments for storing reagents and reacting the sample. The device is configured to detect signals produced from the CRISPR reactions (e.g., optical signals), and thus facilitates a plurality of measurements from a single sample input. A possible application of the device is to perform separate series of amplification and CRISPR reactions to assay a single biological sample for a large number of viruses.
A schematic for the microfluidic chip is depicted in
A depiction of the device is provided in
This example describes an assay for detecting flu viral nucleic acids. The assay is a combination of ambient temperature RT-LAMP amplification and guide nucleic acid driven, programmable nuclease-based detection. LAMP protocols often require strict operating temperatures that are unfeasible for implementation in devices that perform multiple types of reactions. For example, the high temperatures required for some amplification reactions can damage reagents for CRISPR reactions. This example discloses activators for LAMP amplification that are operable at a range of temperatures, including ambient temperatures, that are more suitable for implementation within a device. This example also provides viral lysis buffers containing the LAMP activators, enabling concurrent lysis and amplification upon input of a sample, such as a swab containing nucleic acids associated with the flu.
A variety of potential LAMP activators were tested for LAMP activating capacity and viral lysis buffer compatibility. LAMP activating capacity was evaluated by performing dual LAMP-DETECTR assays in the absence of individual LAMP activators. In these assays, LAMP was performed with three out of four of a buffering agent, an activator, dNTPs, and primer. The DETECTR reactions were performed on buccal swab samples with SEQ ID NO: 37 and the guide nucleic acid (targeting HERC2) given in TABLE 25 below. The DETECTR reactions were monitored by fluorescence over 90 minutes. A separate control assay was performed with all four reagents present during the LAMP amplification. As shown in
This example describes a fully integrated device capable of performing multiple amplification and DETECTR reactions on one input sample. The device contains an inlet port for inserting a sample, an injection-molded cartridge containing reagents for the amplification and DETECTR reactions, a fluidic system for partitioning a sample for multiple reactions, detection components for analyzing the reactions, and hardware for processing the reactions. Inserting a sample into the inlet port seals the sample within the device, preventing the sample and surrounding environment from contamination.
The injection-molded cartridge contains an inlet port for sample insertion. Once the injection-molded cartridge has been prepared with reagents and sealed, a sample can be collected on a swab and inserted into the inlet port. The inlet port is configured so that a swab can be snapped at a break point within the inlet port to fix the sample within the injection-molded cartridge. Once a sample has been fixed in the injection-molded cartridge, the inlet port can be sealed with a hermetic lid.
The sealed injection-molded cartridge (loaded with reagents and a sample) can be inserted into the device, which automates sample preparation and analysis. The device first incubates the sample with 200 μl lysis buffer for 2 minutes. The device meters 20 μl aliquots of the sample into 80 or 180 μl LAMP mastermix for isothermal amplification at 60° C. for 10-60 minutes. 10 μl aliquots of the resulting amplicon are metered into 90 or 190 μl solutions containing DETECTR reagents, and incubated at 37° C. concurrent with real-time excitation and detection at 470 nm and 520 nm. The device collects and transfers this data (e.g., as a radio signal) to computing devices for analysis. The device can perform and detect a large number of sequential and parallel amplification and detection reactions targeting different nucleic acid sequences on a single sample.
This example provides a design for an injection molded cartridge capable of partitioning a sample for separate amplification and DETECTR reactions. The injection-molded cartridge is designed to collect samples from swabs (e.g., buccal swabs). The combinations of distinct amplification and DETECTR reactions allow the sample to be assayed for multiple sequences. For example, the 8 DETECTR reaction could be used to query for 8 separate viruses or 7 viruses and an internal control. The injection-molded cartridge is designed to fit within a device that automates sample and reagent movement, heating, and detection.
This example describes an injection-molded cartridge designed to perform multiple amplification and CRISPR reactions on a single sample. This cartridge has 4 amplification chambers and 8 detection chambers. A single sample will first be diluted in a sample chamber, and then be partitioned between the four amplification chambers. The amplification products from each amplification chamber will be partitioned to two separate detection chambers. Each amplification chamber is transparent so as to allow optical (e.g., fluorescent) monitoring of the CRISPR (e.g., DETECTR) reactions. Each amplification and detection chamber is connected to a unique reagent storage chamber (e.g., an amplification reagent chamber). Some chambers can be loaded with identical reagents, or each chamber can be loaded with different reagents (e.g., amplification reagents and DETECTR reagents targeting different sequences). Thus, the injection-molded cartridge is capable of performing up to 8 unique sequences of amplification and CRISPR reactions on a single input sample.
The injection-molded cartridge is configured to insert into a device capable of controlling sample partition, reagent loading, heating and detection within the cartridge. The cartridge contains multiple valves along with a pneumatic delivery manifold, which collectively allow a device to control the flow, pressure, and temperature in the chambers and fluidic channels within the device. The device can also be equipped with an optical detector (e.g., a fluorimeter) capable of measuring the components of the detection chambers.
As shown from the bottom-up view depicted in panel C, the contents of the amplification reagent chambers can flow into the amplification chambers 101. Mixing is performed by moving the contents back and forth between the two chambers. Once mixing is complete, the samples are completely transferred into the amplification chambers and incubated for a controlled period of time. As is shown in panel D, the inj ection-molded cartridge can be situated over a heating element within the control-device, thus allowing temperature control during the amplification during.
The direction of flow into and out of the amplification chambers is mediated by a slider valve 111. Panel C depicts the slider valve in a first position that connects each amplification reagent chamber to an amplification chamber. Once the amplification reaction is complete, the panel can slide to second and third positions (one of which is depicted in panel E) that allow sample to move from the amplification chambers into metering channels 112. The slider is then capable of adopting a fourth position in which the metering channels overlap with channels 113 that lead to the detection reagent chambers. Thus, the sample is divided into 8 separate components following amplification.
This example covers a detection scheme for fluorescent read-out DETECTR reactions in a multi-chamber cartridge. The cartridge is designed to perform separate DETECTR reactions on separate portions of a sample that have undergone amplification.
This example describes a DETECTR Assay performed on the injection molded cartridge of EXAMPLE 67 using the diode array of EXAMPLE 68. The reagents for the DETECTR assays were loaded directly into the detection chambers. The assays utilized a programmable nuclease with SEQ ID NO: 37, a guide nucleic acid with SEQ ID NO: 256 targeting HERC2 G SNP allele, and a reporter nucleic acid which increased fluorescence response upon cleavage. Four wells contained 5 μM reporter, 150 nM programmable nuclease, 600 nM guide nucleic acid, and 500 pM target nucleic acid. Two wells contained 5 μM reporter, 150 nM programmable nucleic acid, 600 nM guide nucleic, and no target. Two wells contained only buffer. The reporters contained either ATTO 488 or ATTO 594.
This example describes amplification of a target nucleic acid in a viral lysis buffer. The effects of various buffer compositions, reducing agents, and incubation temperatures were tested on amplification of a target nucleic acid. Samples in different buffers were amplified using LAMP amplification, and the resulting fluorescence was measured. Higher fluorescence was indicative of more amplification.
The results of this experiment demonstrated certain buffers were more conducive to LAMP amplification.
This example describes amplification of a target nucleic acid from COVID-19 patient samples in a viral lysis buffer. Samples collected from patients positive for COVID-19 were lysed and amplified in viral lysis buffers with varying components. Target nucleic acids corresponding to the SARS-CoV-2 N gene and RNaseP were amplified using LAMP as described in EXAMPLE 22. Various viral lysis buffer formulations were tested.
While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.
This application is a continuation of International Application No. PCT/US2020/038242, filed Jun. 17, 2020, which claims priority to and benefit from U.S. Provisional Application No.: 62/863,178, filed on June 18, 2019, U.S. Provisional Application No.: 62/879,325, filed on Jul. 26, 2019, U.S. Provisional Application No.: 62/881,809, filed on Aug. 1, 2019, U.S. Provisional Application No.: 62/944,926, filed on Dec. 6, 2019, and U.S. Provisional Application No.: 62/985,850, filed on Mar. 5, 2020, the entire contents of each of which are herein incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62985850 | Mar 2020 | US | |
| 62944926 | Dec 2019 | US | |
| 62881809 | Aug 2019 | US | |
| 62879325 | Jul 2019 | US | |
| 62863178 | Jun 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2020/038242 | Jun 2020 | US |
| Child | 17555236 | US |
