Patent Application
20210207206
Appendix 1 filed herewith further describes certain embodiments of the present technology, and forms part of the original disclosure provided herein.
The present technology relates generally systems, devices, and methods for pathogen detection and characterization. In particular, the present technology provides oligonucleotide ligation assay devices for detecting the presence of one or more mutants within a pathogen.
Nearly 40 million people worldwide are HIV-infected. Effective antiretroviral therapy (ART) suppresses viral replication, which allows HIV-infected individuals to live healthy and productive lives and reduces their risk of transmitting HIV to others. However, the error-prone HIV reverse transcription generates single-base mutants that can confer HIV drug resistance (HIVDR). Mutants at specific locations correlate closely with in vitro susceptibility testing and with clinical treatment failure. Recently, the prevalence of HIV drug resistance (HIVDR) has increased globally and threatens to render some existing drugs ineffective. More specifically, HIVDR to non-nucleoside reverse transcriptase inhibitors (NRTI) used in first-line ART continues to increase and pose a threat to successfully treating and repressing HIV in infected person. For example, persons with transmitted HIVDR may fail to achieve suppression of HIV replication after ART initiation. Additionally, when individuals do not adhere to their daily ART regimen, virus replication may not be suppressed (i.e., treatment failure), and HIVDR variants may be selected.
The present technology provides systems and methods for template dependent ligation. For example, some embodiments of the present technology provide systems and methods for substantially isothermal template dependent ligation for use with an oligonucleotide ligation assay (“OLA”). As will be described in greater detail below, substantially isothermal template dependent ligation can be useful in point of care settings to quickly and accurately detect the presence of certain genetic mutants. For example, the ligation systems and methods disclosed herein can be used to detect the presence of single nucleotide polymorphisms in a target region of an HIV nucleotide sequence that confers HIVDR. By detecting the presence of single nucleotide polymorphisms, the systems and methods provided herein can at least in part provide information on whether a drug regimen given to an HIV infected patient is no longer effective due to drug resistance mutants within the HIV genetic information. The results of the template dependent ligation assays can be interpreted in view of other test results (e.g., viral load testing) to determine other information useful in developing a treatment regimen for an HIV infected patient. For example, the combination of drug resistance and viral load can be used to determine patient adherence to a therapeutic regimen.
The terminology used in the description presented below is intended to be interpreted in its broadest reasonable manner, even though it is being used in conjunction with a detailed description of certain specific embodiments of the present technology. Certain terms may even be emphasized below; however, any terminology intended to be interpreted in any restricted manner will be overtly and specifically defined as such in this Detailed Description section. Additionally, the present technology can include other embodiments that are within the scope of the claims but are not described in detail with respect to
Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present technology. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features or characteristics may be combined in any suitable manner in one or more embodiments.
Reference throughout this specification to relative terms such as, for example, “substantially,” “approximately,” and “about” are used herein to mean the stated value plus or minus 5%.
The headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed present technology.
Testing for HIVDR can identify the appropriate therapeutic option for an HIV-infected patient, including prevention of unnecessary switching of ART to more expensive regimens if treatment is failing due to low adherence rather than HIVDR. HIVDR testing can thus assist clinicians in selecting effective treatments. However, the complexity and cost of genotyping assays limit routine HIVDR testing in resource-limited settings where HIVDR prevalence has reached high levels. Commonly used Sanger sequencing is too expensive ($150-$300 per test) for routine HIVDR testing in many settings. Moreover, slow turnaround times for HIVDR genotypes are common due to the requirement of shipping specimens to centralized laboratories, thereby delaying changes in a therapeutic regimen for patients with a failing regimen.
Low aderence to medication can drastically impair drug efficiency and create opportunities for emerging drug-resistant pathogens. One approach to track how well a patient adheres to their medication is detection of byproducts or metabolites in urine or hair follicles. However, these metabolites may not be directly detectable by a rapid colorimetric method but rather require mass spec-based characterization methods and intensive data analysis. Current public health strategies rely on measuring viral or bacterial loads to indirectly evaluate if a person adheres to their medications. If the viral or bacterial load falls under a certain threshold (depending on the public health strategy), then that means that patient is taking their medications and has benefited from therapies. However, when the viral or bacterial loads are not suppressed, the clinicians provide repetitive counseling to ensure a patient is taking the medication and, if a high load persists, switch treatment. The multiple wait time points between tests can lead to loss to follow up and increased public health risks. This often leads to unnecessarily or delayed switching.
Certain embodiments of the present technology thus provide a synergistic system to gain the insights for an individual's adherence to a therapeutic regimen. For example, the combined use of a pathogen load test (e.g., a viral load and/or bacterial load test) and a drug resistance test can enable primary care clinicians to decide whether a patient needs to switch their treatment. The combined results of pathogen load and resistance testing can yield three potential outcomes that enable clinicians to identify if a person has adhered to their medication, displayed in Table 1.
The present disclosure thus offers synergistic use of pathogen load and drug resistance test results to determine whether a patient adheres to their medication. These two tests can be run independently, simultaneously, and/or sequentially within a certain time frame. For example, the tests can be performed substantially simultaneously such that the test will provide results within a single patient visit (e.g., within 4 hours or less, within 3 hours or less, within 2 hours or less, within 1 hour or less, etc.). In some embodiments, the pathogen load test and the drug resistance test can be integrated into a single OLA device.
Oligonucleotide ligation assays can identify a specific nucleotide sequence in a target strand without the use of radiochemicals, electrophoresis, or centrifugation. In its simplest form, OLAs use at least two probes: a test probe and a common probe. The test probe hybridizes to a first region of the target strand having a sequence complimentary to the test probe, and the common probe hybridizes to a second region of the target strand having a sequence complimentary to the common probe. The first and second regions can be immediately adjacent, or they can be spaced apart by one or more nucleotides. If the probes match the template (e.g., fully complimentary in a region surrounding the portion of the probes to be ligated), a ligase can join the two probes together in a process called ligation. The ligated probes can then be captured and/or detected to signal the presence of a specific nucleotide sequence.
OLAs can be used to detect any potential sequence on a target DNA or RNA strand. To test for a specific sequence, a test probe having a sequence corresponding to the specific sequence can be used. Certain embodiments of the present technology can include first test probes having a first nucleotide sequence comprising a first potential sequence of the target strand, second test probes having a second nucleotide sequence comprising a second potential sequence of the template strand, third test probes having a third nucleotide sequence comprising a third potential sequence of the template strand, etc. While the foregoing description discusses three test probes, the number of probes used can vary based on the number of potential sequences to be detected/tested for. In some embodiments, the first probe may have sequence corresponding to a wild type sequence, the second probe may have a sequence corresponding to a first mutant sequence (e.g., differing from the wild type sequence by a single nucleotide polymorphism), and the third probe may have a sequence corresponding to a second mutant sequence (e.g., differing from the wild type sequence by a single nucleotide polymorphism at a different position than the single nucleotide polymorphism of the first mutant sequence). In other embodiments, the first, second, and third probes have sequences corresponding to first, second, and third mutants, respectively.
As will be described in greater detail herein, the test probes can further include capture molecules to facilitate retention of ligated probes. For example, the first test probe may include a first hapten, a first immuno-tag, a first nucleotide barcode (e.g., a first unique DNA. PNA, LNA, or pDNA sequence), or other first molecule for selectively retaining the first test probe. The second test probe can include a second hapten, a second immuno-tag, a second nucleotide barcode (e.g., a second unique DNA, PNA, LNA, or pDNA sequence), or other second molecule for selectively retaining the second test probe. The third test probe may include a third hapten, a third immune-tag, a third nucleotide barcode (e.g., a third unique DNA, PNA, LNA, or pDNA sequence), or other third molecule for selectively retaining the third test probe. In some embodiments, the first, second, and third capture molecules are different.
The OLAs can also include common probes. The common probes can have a nucleic acid sequence substantially complimentary or complimentary to a second region of the template strand adjacent to the first region. Thus, when a common probe and a test probe are hybridized to the same template strand, the common probe and the test probe are adjacent to one another, and ligase (e.g., Taq DNA ligase) can ligate the common probe and the test probe by catalyzing the formation of a phosphodiester bond between a first end region of a test probe (e.g., the 5′ end of the test probe) and a first end region of the common probe (e.g., the phosphorylated 3′ end of the common end). Notably, while the first, second, and third test probes may compete to hybridize to the first region, the first, second, and third test probes will only be ligated to the common probe if their sequence is complimentary to the sequence of the template strand along the end region near the end that will be ligated to the common probe. In some embodiments, the test probe and the common probe can be immediately adjacent to another when hybridized to the template strand. In some embodiments, the test probe and the common probe can be spaced apart by one or more nucleotides when hybridized to the template strand. In some embodiments, the common probes can include a reporter molecule for providing a signal. For example, the common probes may include fluorescein, biotin, a hapten, or another molecule that can provide a detectable signal. In other embodiments, the common probes do not include reporter molecules.
A number of different ligated probes can form between the plurality of test probes and the common probes. For example, the ligated probes can include first ligated probes comprising the first test probe ligated to the common probe, second ligated probes comprising the second test probe ligated to the common probe, third ligated probes comprising the third test probe ligated to the common probe, etc. As will be discussed in more detail below, the first, second, and third ligated probes can be selectively captured and/or detected in first, second, and third regions of a detection zone to identify and quantify the presence of specific nucleic acid sequences in the target strand.
As one skilled in the art will appreciate, the probes (e.g., the wild type probes, the mutant probes, and/or the common probes) may not be perfectly complementary to the full region of the template strand to which they correspond. For example, certain target pathogens (e.g., HIV RNA) are highly variable. Accordingly, the probes can be made longer than required for ligation purposes and/or the assay can be run significantly below the probe melting temperature so that the probes can bind the template strand despite a few mismatches. Nonetheless, the OLA assays are still able to identify specific mutations (e.g., single nucleotide polymorphisms) due to the specificity of the ligase, which requires the bases near the ligation site to be perfect matches. For example, when the 5′ end of the test probe is to be ligated to the 3′ end of the common probe, ligation will only occur if the bases near the 5′ end region of the test probe and the 3′ end region of the common probe are perfectly complementary to the template strand. Likewise, when the 3′ end of the test probe is to be ligated to the 5′ end of the common probe, ligation will only occur if the bases near the 3′ end region of the test probe and the 5′ end region of the common probe are perfectly complementary to the template strand. Accordingly, the specific sequence of interest (e.g., the single nucleotide polymorphism) can be at or near the end of the test strand that will be ligated to the common strand to enable precise ligation and detection of specific sequences. For example, when the 5′ end of the test strand is to be ligated to the 3′ end of the common strand, the 5′ end region of the test strand can have the nucleotide corresponding to the single nucleotide polymorphism.
As one skilled in the art will appreciate, OLAs can be used in a variety of applications. The probe sequences will be selected based on the specific sequences the target strand is being tested for. When testing for HIVDR, for example, an OLA assay may test for a number of different mutations, including, for example, K65R, K103N, V106M, Y181C, M184V, G190A, Q148H, Q148R, Q148K, N155H. and/or R263K. Exemplary probes used to detect these mutant sequences, as well as associated wild type probes and common probes, are provided as SEQ. ID NOs. 1-35 in Table 2.
The present technology can also be used to test for the presence of certain human leukocyte antigen (HLA) genotypes. Certain HLA genotypes (e.g., HLA b57) indicate a patient may have a hypersensitivity to certain anti-retroviral therapies (e.g., Abacavir). Exemplary probes for HLA genotyping are provided as SEQ ID NOs. 36-38 in Table 3.
In certain embodiments of the present technology, human betoglobin can be used as a control for various OLA tests. Sequences for human betoglobin probes are provided as SEQ. ID. NOs. 39 and 40 in Table 4.
As will be discussed in greater detail herein. OLA typically requires target strand of RNA or DNA to be amplified prior to the ligation assay. Accordingly, the present technology also provides primers corresponding to select regions of interest on target strands. For example, SEQ ID. NOs. 41-46 set forth sequences for primers used to amplify the HIV-integrase region, the HLA B57 region, and human betoglobin, as provided in Table 5.
The specific mutations, probes, and primers are provided herein as exemplary applications of the present technology. As one skilled in the art will appreciate from the disclosure, the present technology provides systems and methods that can test for a wide variety of mutations in various applications. Accordingly, the present technology is not limited to testing for the specific mutations or using the specific probes or primers disclosed herein.
Certain embodiments of the present technology provide systems, devices, and methods for template dependent ligation to identify specific nucleic acid sequences in a region of interest on a target strand of DNA or RNA. For example, template dependent ligation can be used to identify the presence or absence of single nucleotide polymorphisms in a region of interest in a target strand. In certain embodiments, for example, template dependent ligation can be used to detect mutants within HIV genetic information.
Either before or after the heating step, one or more probes (including test probes 128 and common probes 130) can be mixed with the DNA amplicons 105. The temperature can be reduced to a temperature that facilitates hybridization between the probes 128, 130 and the template strand 105a (e g., between about 35-45 degrees Celsius). As will be discussed in more detail herein, the probes 128, 130 hybridized to adjacent regions on the template strand 105a can be ligated and captured to detect the presence of single nucleotide polymorphisms. However, while the temperature decrease can enable the template strands 105a to anneal to the probes 128,130, a portion of the template strands 105a will re-hybridize to the non-template strand 105b rather than anneal to the probes 128, 130. Thus, there is a binding competition between the non-template strand 105b and the probes 128,130, resulting in a low ligation efficiency (about 2%). As a result, this traditional process is inefficient at producing sufficient hybridization between the template strand 105a and the probes 728,730, and thus is inefficient at producing ligated probes 731. To account for this inefficiency, the steps of heating and cooling are repeated in a process called thermal cycling. The number of cycles can vary on the quantity of ligated probes 131 needed, but a typical assay will require at least 10 cycles. After a sufficient amount of ligated probes have been formed, the ligated probes can be captured and detected through a variety of techniques. For example, the ligated probes can be detected through techniques known in the art such as enzyme-linked immunosorbent assays (ELISA).
As discussed above, the double-stranded DNA amplicons can be heated to dissociate the template strand and non-template strand to facilitate subsequent template dependent ligation. However, because the dissociated non-template strand competes with the probes for annealing to the template strand, the process of heating the amplified sample to promote dissociation and cooling the amplified sample to promote hybridization and ligation must be continually repeated in a process known as thermal cycling Certain embodiments of the present technology provide systems and methods for template dependent ligation that can negate the need for thermal cycling and therefore increase the efficiency of template dependent ligation.
Increasing the quantity and/or the concentration of the template strand to provide the template strand in single stranded form for template dependent ligation can be achieved through a variety of techniques. Such techniques can include pre-amplification techniques, amplification techniques, and post-amplification techniques (collectively referred to as “pre-ligation techniques”). In some embodiments, the pre-ligation techniques can be used after polymerase chain reaction or other amplification methods. As one of skill in the art will appreciate from the disclosure herein, these techniques can be used alone or in combination, and are used to prepare a sample for template dependent ligation.
Pre-amplification techniques involve treating the biological sample before amplification such that, following amplification, there is an increased concentration of the template strand versus the non-template strand. Pre-amplification techniques include treating the pre-amplified sample with an enzyme to digest one strand of the pre-amplified double-stranded DNA. For example, enzymes such as lambda exonuclease can selectively digest one strand of the double-stranded DNA (e.g., the phosphorylated 5′ end of a modified amplicon) to generate single stranded DNA template prior to amplification.
Amplification techniques include providing a first primer (e.g., a reverse primer) for amplifying a first strand at a higher concentration than a second primer (e.g., a forward primer) for amplifying a second strand to facilitate asymmetrical amplification. Using a first primer at a higher concentration than a second primer can generate partially single stranded DNA. As a result, after amplification, there is no need for heat treatment to dissociate the double-stranded DNA because there is sufficient single-stranded DNA. For example, in some embodiments, the primer for amplifying the template strand may be provided in higher concentrations than the primer for amplifying the non-template strand such that the template strand is provided in singe-stranded form following amplification.
Post-amplification techniques include techniques for generating single-stranded DNA amplicons after amplification of the target strand. For example, double-stranded DNA amplicons may be treated via one or more of the following techniques: (1) a heating method to induce strand breathing; (2) a strand displacement and exchange method; (3) enzymatic mediated strand invading; (4) enzymatic strand digestion; or (5) chemical dissociation methods. In the strand breathing technique, the double-stranded DNA amplicons can be incubated with the probes at temperatures about 5-10 degrees lower or higher than the melting temperature of the probes. While this temperature may not cause the double-stranded DNA to fully dissociate into single-stranded form, the increased temperature can cause the strands to partially dissociate in a process known as strand breathing. This slight dissociation can enable the probes to compete with the non-template strand for hybridization with the template strand. In the strand displacement and exchange technique, a single-stranded competing oligonucleotide can be mixed with the amplicons. If the single-stranded competing oligonucleotide forms a more thermodynamically stable double-stranded DNA duplex with the non-template strand, the template strand will dissociate from the non-template strand in favor of the more thermodynamically stable double-stranded DNA duplex. In the enzymatic mediated strand invading technique, recombinase enzymes can bind to single-stranded DNA or free single stranded probe and insert the probe into the duplex of the DNA template. In another example of enzymatic mediated strand invading, enzymes such as heat liable and/or heat stable helicase can be used to de-hybridize the double-stranded DNA and allow for the kinetically favorable probes to bind the de-hybridized template and get ligated. In enzymatic strand digestion, an enzyme that selectively digests one strand of the double-stranded DNA can be added to the sample. For example, lambda exonuclease can selectively digest one strand of the double-stranded DNA (e.g., the phosphorylated 5′ end of a modified amplicon) to provide the template strand in single stranded form. In chemical dissociation methods, the environment including the amplicons can be treated to facilitate dissociation between the double-stranded amplicons. For example, a strong base (e.g., sodium hydroxide) can be added. In other embodiments, a salt concentration of the environment can be reduced.
Following amplification, probes can be combined with the amplified sample, and the single stranded amplicons can anneal to the probes (process step 304). The probes can include a first test probe having a first nucleotide sequence and a common probe having a second nucleotide sequence. The method 300 can continue by ligating at least a portion of the probes via template dependent ligation based on the single stranded amplicons to generate ligated probes (process step 306). The ligated probes can comprise first ligated probes having the first test probe ligated to the common probe. Different or constant probe concentrations can be used to perform ligation on single-stranded amplicons. The method 300 continues by detecting the ligated probes, wherein detecting the first ligated probe indicates the target strand included the first nucleotide sequence (process step 308). As noted above, the method 300 does not require thermal cycling, and can proceed to the step of capturing and/or detecting the ligated probes without the need for repeated cycles of heating and cooling. In some embodiments, the steps of asymmetrically amplifying, annealing, and ligating can be performed at temperatures at or below 70 degrees Celsius. In some embodiments, the steps of asymmetrically amplifying, annealing, and ligating can be performed at temperatures between 35-45 degrees Celsius. In some embodiments the step of asymmetrically amplifying is performed at 37 degrees Celsius and the step of ligating is performed at 40 degrees Celsius. In some embodiments, the steps of asymmetrically amplifying, annealing, and ligating are performed in a substantially isothermal environment. In some embodiments, the temperature is determined by the operating temperature range of the ligase (e.g., between about 37-65 degrees Celsius for Taq DNA Ligase).
Method 300 can further include second test probes for identifying second nucleotide sequences in the target strand. For example, the second test probes can have a second nucleotide sequence corresponding to a second potential sequence of the target strand. The second test probe can be ligated to the common probe if the second nuclotide sequence is present on the target strand. Accordingly, the second ligated probes can be detected to determine whether the target strand includes the second nucleotide sequence. As one skilled in the art will appreciate from the disclosure herein, additional test probes corresponding to additional potential sequences of the target strand can be added to determine whether the target strand has the potential sequence.
The method 350 can continue by annealing one or more of the single stranded amplicons to one or more of a plurality of probes (process step 354). The plurality of probes can include (1) wild type probes having a nucleotide sequence comprising a wild type sequence of the target strand, (2) at least one mutant probe having a nucleotide sequence comprising at least one potential mutant sequence of the target strand, the at least one potential mutant sequence including a single nucleotide polymorphism compared to the wild type sequence, and (3) common probes. In some embodiments, the plurality of probes could include additional mutant probes for testing other potential mutant sequences. In some embodiments, the method 350 can be performed without the wild type probe (i.e., using only mutant probes and common probes). The method 350 continues by ligating individual common probes to individual wild type probes and/or individual mutated probes to form a plurality of ligated probes each comprising a wild type probe and a common probe or a mutant probe and a common probe (process step 356). Ligation can occur when a wild type probe or a mutant probe is hybridized to a complimentary sequence along the single stranded amplicons. The method 350 can continue by capturing the ligated probes comprising the wild type probe and the common probe in a first region and capturing the ligated probes comprising the mutant probe and the common probe in a second region (process steps 358, 360). The method 350 can further include displaying the captured ligated probes (process step 362).
The present technology also provides devices architectures, systems, and devices for facilitating template-dependent ligation. For example, certain embodiments of the present technology provide disposable oligonucleotide ligation assay (OLA) devices. The OLA devices can be capable of performing the OLA workflows described herein and can be used to diagnose and characterize various infections. For example, the OLA devices described herein can be used to diagnose HIV and to determine whether an infected patient has drug resistant HIV. The OLA devices can also provide information for determining a patient's adherence to a therapeutic regimen. As will be described in detail with respect to
The sample port 504 can receive a container holding the biological sample (e.g., collection tubes). In some embodiments, the sample port 504 mates with an external surface of the container such that a bottom portion of the container is inside the casing 502 and fluidly isolated from the external environment. By fluidly isolating the bottom portion of the container from the external environment, the sample port 504 can reduce contamination from the device 500. For example, in some embodiments, the biological sample is amplified before delivering the sample to the device. In such embodiments, the sample port reduces potential contamination. To facilitate transfer between the container and the interior of the device 500, the sample port 504 can include a piercing element (not shown). The piercing element can be any element (e.g., a pin, a blade, etc.) suitable for piercing the bottom portion of the container holding the biological sample to release the biological sample onto the paper flow membrane. In other embodiments, the sample port 504 does not include a piercing element, and the biological sample is pipetted or otherwise transferred to the sample port 504.
The buffer port 506 is positioned adjacent a dried reagent pad (not shown) contained within the casing 502. The buffer port 506 is in fluid connection with the dried reagent pad such that, when liquid is added to the buffer port 506, the dried reagent pad becomes rehydrated. As will be discussed in greater detail with respect to
The pathogen load detection zone 508 signals the presence of a target pathogen (e.g., HIV) in the biological sample via, for example, color bands as described above with respect to
The drug resistance detection zone 510 signals whether the biological sample contains one or more sequences indicating resistance. The drug resistance detection zone 510 can be divided into a plurality of regions, with each region corresponding to a specific mutant. For example, if the device 500 is testing a biological sample for drug resistant HIV, the drug resistance detection zone 510 may have a plurality of regions 510a-f, with each region signaling the presence (or absence) of a given mutant. For example, the regions may signal the presence of the following mutants: K65R, K103N, V106M, Y181C, M184V, and G190A.
As illustrated in
The reagent pad 512 can comprise a plurality of dried reagents 514a-e (e.g., lyophilized reagents). For example, the plurality of dried reagents 514a-e can comprise dried reagents beads prepared using the systems and methods disclosed herein with respect to
The lateral flow membrane can be divided into a first zone 520a, a second zone 520b, and a third zone 520c. The first zone 520a can receive the biological sample from the sample port 504. As described in greater detail below with respect to
The test probes 528 can also include a second region 529b having a unique nucleotide sequence. For example, the first test probes can include a second region having a first unique nucleotide sequence, the second test probes can include a second region having a second unique nucleotide sequence, and the third test probes can include a second region having a third unique nucleotide sequence. The first unique nucleotide sequence, the second unique nucleotide sequence, and the third unique nucleotide sequences can be DNA barcodes that enable the first test probes, the second test probes, and the third test probes to be captured in different regions in the third zone 520c. The DNA barcodes can be computationally-designed and thus show high sensitivity (about 100% capture), low cross-reactivity, and tolerance to temperature variation from about 25-4 degrees Celsius. The barcodes can include DNA or DNA derivatives such as PNA, LNA, and/or pDNA.
The test probes 528 and/or the common probes 530 can hybridize to complementary regions along the retained template strands 805a. Ligase (e.g., Taq DNA Ligase) can ligate individual test probes 528 to individual common probes 530 when the individual test probe 528 and the individual common probe 530 are hybridized to adjacent regions of the template strand 805a to form ligated probes 531. As described herein, test probes 528 will only be ligated to common probes 530 when the test probes 528 are complementary to the region of the template strand 805a the 5′ end region of the test probe 528 is hybridized to. This process can continue for the duration of the ligation incubation period (e.g., about 5 minutes). Following the ligation incubation period, the device 500 can be heated to temperatures of about 70 degrees Celsius to denature the ligated probes and release them from the amplicons. In other embodiments, the device 500 can be heated to temperatures of about 70 degrees Celsius or higher (e.g., about 10 degrees Celsius higher than the melting temperature of the probes) and/or a wash buffer (e.g., an alkaline buffer) can be used to separate the ligated probes from the amplicons. In some embodiments, the probes can be released from the amplicons through competing hybridization. For example, invader strands comprising an identical or substantially identical sequence to the footprint of the ligated probes and a sticky end of about 6 nucleotides or more can be added to the lateral flow membrane. The sticky ends of the invader strands can bind to the template strands and undergo toehold mediated strand displacement, where the resultant invader strand-template strand duplexes are more thermodynamically stable than the test probes-template duplexes. This also reduces the likelihood of detecting a false positive in the drug resistance detection zone by reducing the likelihood that an unligated test probe and an unligated common probe will remain hybridized to the same template strand in the detection zone. In some embodiments, the sticky end can also hybridize to the strand containing the region complementary to the sticky end and the other region complementary to the probe itself. This enables unligated probes to be free from the target. In embodiments that utilize heat to denature the ligated probes, heat can be provided by a number of sources, including an on-board circuit and battery in the device 500 or a reusable heater with slots for the device 500. If a reusable heater is utilized, the same heater used for a VF test can provide ports for the device 500 (with switches to detect device inserted, status lights, communication with software, etc.). In embodiments that use competing hybridization, competing oligonucleotides can be provided to promote dissociation between the ligated probes and the template strands.
Following the ligation incubation period, the reagent pad 512 can release the fourth reagent 514c, as illustrated in
Following release of the fourth reagent 514c, the reagent pad 512 can release the fifth reagent 514d. The fifth reagent 514d can include a substrate that can interact with the common probes and cause the common probes to emit a signal (e.g., a color signal). For example, the substrate can interact with the labeling enzymes to cause the labeling enzymes to emit a signal. In other embodiments, the substrate can interact directly with the common probe and cause the common probe to emit a signal. The signals indicate a “positive” result for the given region the signal is associated with.
The entire workflow described about with respect to
The intensities of lines (or dots) reporting the presence of specific mutants from the OLA device described herein can be quantified and reported as a value that reflect the loads of single nucleotide polymorphisms detected. The higher the intensity of the line, the more prevalent the corresponding single nucleotide polymorphism is in the biological sample. Thus, signal intensities can be used to gain further information to characterize an individual's pathogen load. For example, the loads of one or more single nucleotide polymorphisms detected on lateral flow tests described herein can be analyzed and converted into one or more numbers classifying the intensity of the signal. The numbers can then be used by physicians and care givers to select an appropriate therapeutic regiment for the patient.
In some embodiments, a computer can analyze the signal and quantify the intensity of the signals via an algorithm. Exemplary algorithms can, for example, convert the detection results of a set of one or more SNP the signal obtained from lines or other shapes on the lateral flow tests or other substrates to one or more numbers, and/or convert the set of one or more numbers obtained from the detection zones into the predictive output such as but not limited to resistance or susceptibility to a particular or a combination of drug agents.
A user can add a sample collection tube 934 containing a biological sample into the sample port 904. The sample port 904 can contain a piercing element that punctures the bottom of the collection tube 934 to release the biological sample onto the lateral flow membrane 920. The user can add a rehydration buffer 907 to the buffer port 906 to rehydrate the plurality of dried reagent pads 914a-e. The user can then pull the tab 918 separating the plurality of rehydrated reagents and the lateral flow membrane 920 to release the plurality of rehydrated reagents on the lateral flow membrane. As described above and further illustrated in
As one skilled in the art will appreciate from the foregoing, a number of modifications could be made to the workflows and devices described with respect to
While the OLA devices described with respect to
The present technology provides systems, devices, and associated methods for collecting and preparing a biological sample for use with systems and methods described herein. For example, before the amplification and ligation methods described herein can be performed, a biological sample containing target RNA or DNA must be obtained from an infected patient. The biological sample must also be treated such that the target RNA or DNA is available for amplification and template dependent ligation (e.g., lysed). Thus, the present technology provides an integrated sample collector and preparation device.
The integrated sample collector and preparation devices provide numerous advantages over traditional sample collection and preparation devices and methods. For example, the integrated sample collector and preparation device 1000 can streamline sample collection and preparation to increase efficiency—a typical workflow of sample collection and preparation using the syringe can take about 2 minutes or less. In addition, the integrated sample collector and preparation device 1000 can be used by personal with little to no user training. The user will simply be collecting blood usual. Moreover, users will handle lysed plasma, which is less infectious than whole blood. As one skilled in the art will appreciate, the integrated sample collector and preparation device can be altered for use with various sample collection techniques. For example, the integrated sample collector and preparation device can be used with various methods for collecting a blood sample, including venipuncture and/or finger sticks.
Select embodiments of the present technology provide sample collecting tubes for transferring a biological sample from a sample collection device (e.g., the integrated sample collector and preparation device 1000) to an OLA device (e.g., the devices described herein with respect to
The container 1102 can include a bottom portion 1106 for delivering the biological sample to the OLA device to reduce the likelihood of contamination. For example, the bottom portion 1106 can include a puncturable material. The puncturable material can include, for example, aluminum foil, wax, or any other suitable material that provides a sealed membrane that can be punctured. When the collecting tube 1100 is placed within a sample receiving port 1104 of an OLA device, the puncturable material of the bottom portion 1106 can be punctured to release the biological sample contained within the container 1102 onto the OLA device. As discussed above with respect to
The capture method can be compatible with downstream amplification. For example, the capture method was further validated using a low (pre-amplification) level concentration of synthetic 80-base HIV pol gene (104 copies).
For all-in-one drug resistance and viral load test. RNA in plasma is the clinically accepted specimen. The capture system described herein is also applicable for HIV RNA.
The present technology also provides systems and methods for amplifying the target strand on a lateral flow membrane.
As discussed above, the number of immobilized reverse primers 1424 can be fixed to enable primer-limited amplification. Primer-limited amplification can enable quantification of a drug resistance mutant percentage. Capturing the target strand for amplification on immobilized reverse primers also enables incompatible steps to be performed sequentially on the same device since fluid is washed away between reaction steps (e.g., between amplification and ligation). The reactions can also be spatially separated if needed, for example, to avoid probe overlap. All non-overlapping probes may be detected in one strip and overlapping probes may be reacted sequentially or split for parallel reaction. In addition, embodiments of the device may be made for different drug regimens, (e g., a patient transitioning to TDF+3TC+DTG may need RT (K65R, M184V) and DTG (N148H/R/K, N155H, R263K) in a single test).
Reverse primers can be immobilized on the lateral flow membrane by, for example, UV crosslinking of T20 tails82 or by binding biotinylated primers to immobilized streptavidin. A typical test line on nitrocellulose95 (0.1 cm thick, 1 cm wide) can bind approximately 10{circumflex over ( )}13 streptavidin molecules (50-200 ug/cm2 for IgG95), and approximately 10{circumflex over ( )}9 molecules can be seen by eye with gold labels. Thus, immobilizing 10{circumflex over ( )}12 primers (near 10{circumflex over ( )}11/cm2) is well below the binding capacity and would generate 50×DNA required to be detected if amplification and ligation were 100% efficient and sample was 100% MUT (the aim is to detect approximately 10% MUT; this would require approximately 1% combined efficiency to give detectable product; thus, there is large excess capacity and room for inefficiencies). Amplification can be carried out according to the steps detailed with respect to
Select embodiments of the present technology provide systems and methods for high-throughput dried reagent fabrication. In some embodiments, dried reagents fabricated via the processes described herein can be incorporated into the OLA devices described with respect to
Current processes for fabricating dried reagents take place in an industrial setting and require expensive equipment to be used to release uniformly-sized droplets containing reagent materials into liquid nitrogen for rapid freezing. For small-scale production or situations in which the expensive equipment is not accessible, technicians are required to stand for several hours to produce a large quantity (e.g., one hundred) reagent beads made by releasing the reagents from pipette tips into liquid nitrogen in a drop-wise manner. There are number of challenges associated with this approach, including: (1) the fluid will burst into small droplets if the droplets are released too fast, and (2) the fluid in the tips will freeze if the droplets are released too slowly.
The present technology provides systems and methods for making dried reagent beads.
Once the reagents and/or preservatives are loaded into the reservoirs 1552, the malleable material substrate 1550 can be immersed into liquid nitrogen 1554 to cause rapid freezing of the reagents/preservatives. In other embodiments, the malleable material substrate 1550 and reagents 1560 are frozen via exposure to dry ice. The malleable material substrate 1550 can fracture when submerged into the liquid nitrogen 1554. For example,
Select embodiments of the present technology include point of care kits for pathogen load testing and drug resistance detection. For example, an HIV viral load detection and drug resistance detection kit may include an OLA device, an integrated sample collector and preparation device, and sample collecting tubes as described herein. Such kits could be deliverable to point of care settings to provide rapid, low-cost HIV testing, and to enable efficacious treatment to HIV-infected patients.
1. A method for detecting a mutation in a target strand of DNA or RNA, the method comprising:
2. The method of example 1 wherein ligating the portion of the plurality of probes to form the first ligated probe comprises:
3. The method of example 2 wherein the first region and the second region are immediately adjacent.
4. The method of example 2 wherein the first region and the second region are spaced apart by one or more nucleotides.
5. The method of example 1 wherein the first nucleotide sequence corresponds to a mutant sequence of the target strand.
6. The method of example 1 wherein the first nucleotide sequence includes a single nucleotide polymorphism positioned at a first end region of the first test probe.
7. The method of example 1 wherein the plurality of probes further includes a second test probe having a second nucleotide sequence.
8. The method of example 7 wherein the ligated probes further include second ligated probes comprising the second test probe ligated to the common probe.
9. The method of example 8 wherein ligating the portion of the plurality of probes to form the second ligated probe comprises:
10. The method of example 7 wherein the second nucleotide sequence differs from the first nucleotide sequence by a single nucleotide polymorphism.
11. The method of example 7 wherein the first nucleotide sequence comprises a wild type nucleotide sequence and the second nucleotide sequence comprises a mutant of the wild type sequence.
12. The method of example 1 wherein the common probes each include one or more capture molecules, one or more reporter molecules, one or more fluorescent molecules, or one or more quencher molecules.
13. The method of example 1 wherein amplifying the target strand comprises asymmetrically amplifying the target strand to generate an excess of single stranded amplicons.
14. The method of example 13 wherein asymmetrically amplifying the target strand comprises providing one of either a forward primer or a reverse primer in a higher concentration during amplification.
15. The method of example 1 further comprising treating the amplicons to enable the amplicons to anneal to the plurality of probes.
16. The method of example 15 wherein treating the amplicons comprises heating the amplicons at a temperature of about 70-90 degrees Celsius to denature the amplicons.
17. The method of example 15 wherein treating the amplicons comprises providing an enzyme to digest one strand of the amplicons.
18. The method of example 15 wherein treating the amplicons comprises providing an enzyme that inserts one or more of the plurality of probes into the amplicons.
19. The method of example 15 wherein treating the amplicons comprises altering an environment containing the amplicons to promote de-hybridization, and wherein altering the environment comprises adding a strong base and/or reducing a salt content.
20. The method of example 15 wherein treating the amplicons comprises providing a competing oligonucleotide configured to bind to one strand of the amplicons.
21. The method of example 1 wherein the steps of amplifying, annealing, and ligating are performed at temperatures at or below about 70 degrees Celsius.
22. The method of example 1 wherein the steps of amplifying, annealing, and ligating are performed at temperatures between about 35 degrees Celsius and 45 degrees Celsius.
23. The method of example 1 wherein the steps of amplifying, annealing, and ligating are performed in a substantially isothermal environment.
24. A method for identifying drug-resistant HIV, comprising.
25. The method of example 24 wherein the wild type probe has a nucleotide sequence corresponding to one of SEQ ID NOs. 1, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32.
26. The method of example 24 wherein the mutant probe has a nucleotide sequence corresponding to one of SEQ ID NOs. 2, 6, 9, 12, 15, or 18, 21, 24, 27, 30, or 33.
27. The method of example 24 wherein amplifying the target strand comprises asymmetrically amplifying the target strand to generate an excess of single stranded amplicons.
28. The method of example 27 wherein asymmetrically amplifying the target strand comprises providing one of either a forward primer or a reverse primer in a higher concentration during amplification.
29. The method of example 24 further comprising treating the amplicons to enable the amplicons to anneal to the plurality of probes.
30. The method of example 24 wherein treating the amplicons comprises heating the amplicons at a temperature of about 70-90 degrees Celsius to denature the amplicons.
31. The method of example 24 wherein treating the amplicons comprises providing an enzyme to digest one strand of the amplicons.
32. The method of example 24 wherein treating the amplicons comprises providing an enzyme that inserts one or more of the plurality of probes into the amplicons.
33. The method of example 24 wherein treating the amplicons comprises altering an environment containing the amplicons to promote de-hybridization, and wherein altering the environment comprises adding a strong base and/or reducing a salt content.
34. The method of example 24 wherein treating the amplicons comprises providing a competing oligonucleotide configured to bind to one strand of the amplicons.
35. The method of example 24 wherein the steps of amplifying, annealing, and ligating are performed at temperatures at or below about 70 degrees Celsius.
36. The method of example 24 wherein the steps of amplifying, annealing, and ligating are performed at temperatures between about 35 degrees Celsius and 45 degrees Celsius.
37. The method of example 24 wherein the steps of amplifying, annealing, and ligating are performed in a substantially isothermal environment.
38. A device for identifying mutants in a target strand of DNA or RNA, the device comprising:
39. The device of example 38, further comprising an absorbent pad spaced apart from the dehydrated reagent pad by a length of the lateral flow membrane, wherein the absorbent pad is configured to facilitate fluid flow through the length of the lateral flow membrane.
40. The device of example 38 wherein the device is configured to automatically and sequentially deliver the plurality of dried reagents following rehydration of the reagent pad.
41. The device of example 38 wherein the plurality of dried reagents includes a first reagent for use in the amplification zone and a second reagent for use in the ligation zone, and wherein, after rehydration, the first reagent flows through the lateral flow membrane before the second reagent flows through the lateral flow membrane.
42. The device of example 41 wherein the dried reagent pad is configured such that the release of the second reagent is delayed for a period of time following the release of the first reagent to provide an amplification incubation period.
43. The device of example 42, further comprising a tab configured to control the release of the second reagent, wherein the tab is transitionable between a first position in which the second reagent is retained in the reagent pad and a second position in which the second reagent is released onto the lateral flow membrane.
44. The device of example 38 wherein the plurality of dried reagents include forward primers and reverse primers, and wherein, when the dried reagents are rehydrated, the forward primers and the reverse primers flow through a portion of the lateral flow membrane to the amplification zone.
45. The device of example 44 wherein the forward primers have a first concentration in the dried reagent and the reverse primers have a second concentration in the dried reagent, and wherein the first concentration differs from the second concentration.
46. The device of example 38 wherein the plurality of dried reagents include a plurality of probes, and wherein, when the dried reagents are rehydrated, the plurality of probes flow through a portion of the lateral flow membrane to the ligation zone.
47. The device of example 46 wherein the plurality of probes includes:
48. The device of example 47 wherein the one or more common probes are configured to be ligated to the mutant probes in the ligation zone.
49. The device of example 47 wherein the plurality of probes further includes wild type probes having a nucleotide sequence comprising a wild type sequence of the target strand.
50. The device of example 49 wherein the one or more common probes are configured to be ligated to either the wild type probes or the mutant probes in the ligation zone.
51. The device of example 38 wherein the amplification zone includes a plurality of immobilized primers at least partially complimentary to the target strand.
52. The device of example 38 wherein the detection zone includes a plurality of regions, and wherein each region of the plurality of regions has a unique capture molecule immobilized on the lateral flow membrane.
53. The device of example 52 wherein each of the plurality of regions corresponds to a different specific sequence of the target strand, wherein the different specific sequences include one or more mutant sequences.
54. The device of example 53 wherein the sample port includes a piercing element configured to pierce a housing containing the biological sample to release the biological sample onto the lateral flow membrane.
55. The device of example 38 wherein the device is configured for isothermal amplification and/or isothermal ligation.
56. A device for identifying drug resistant HIV, the device comprising:
57. The device of example 56 wherein the amplification and ligation zone includes a first region configured to capture the target strand and a second region configured to capture single stranded amplicons.
58. The device of example 57 wherein the first region of the amplification and ligation zone is configured to facilitate amplification of the target strand and the second region of the amplification and ligation zone is configured to facilitate ligation of the plurality of probes.
59. The device of example 56 wherein the amplification and ligation zone include a first region configured to capture the target strand, and wherein the first region is configured to facilitate amplification of the target strand and ligation of the plurality of probes.
60. The device of example 56 wherein ligation occurs when (a) the mutant probe is hybridized to a first region of the amplicon complimentary to the mutant probe, and (b) the common probe is hybridized to a second region of the amplicon complimentary to the common probe.
61. The device of example 60 wherein the first region and second region of the amplicon are immediately adjacent.
62. The device of example 60 wherein the first region and the second region of the amplicon are spaced apart one or more nucleotides.
63. The device of example 56 wherein the device is configured for isothermal amplification and ligation.
64. The device of example 56 wherein the common probes include a reporter molecule.
65. The device of example 56 wherein the mutant probes are first mutant probes corresponding to a first potential mutation, the mutant probes further comprising second mutant probes corresponding to a second potential mutation.
66. The device of example 65 wherein the plurality of ligated probes include first ligated probes comprising the first mutant probe and the common probe and/or second ligated probes comprising the second mutant probe and the common probe.
67. The device of example 66 wherein the detection zone includes a first region configured to detect the first ligated probes and a second region configured to detect the second ligated probes.
68. The device of example 67 wherein—
69. The device of example 68 wherein the first region of the detection zone includes immobilized capture molecules comprising a first nucleotide sequence substantially complimentary to the first DNA barcode, and wherein the second region of the third zone includes immobilized capture molecules comprising a second nucleotide sequence substantially complimentary to the second DNA barcode.
70. The device of example 67 wherein the first mutant probes include a first immuno-tag and the second mutant probes include a second immuno-tag, and wherein the first immuno-tag and the second immuno-tag are different.
71. The device of example 70 wherein the first region of the detection zone includes immobilized antibodies configured to bind the first immuno-tag, and wherein the second region of the third zone includes immobilized antibodies configured to bind the second immuno-tag.
72. The device of example 56 wherein the dried reagents include the mutant probes and the common probes.
73. The device of example 56 wherein the dried reagents include reverse primers and/or forward primers.
74. The device of example 56 wherein the amplification and ligation zone includes immobilized reverse primers and/or immobilized forward primers.
75. The device of example 56, further comprising a sample port configured to receive a biological sample including the target strand.
76. The device of example 75 wherein the sample port is fluidly coupled to the lateral flow membrane and is configured to receive a container housing the biological sample, and wherein the sample port includes a piercing element configured to pierce the container to release the biological sample onto the lateral flow membrane.
77. The device of example 75 wherein the sample port is configured to deliver the biological sample to the amplification and ligation zone.
78. The device of example 56 wherein the at least one potential mutant sequence includes a single nucleotide polymorphism compared to a wild type sequence.
79. The device of example 56 wherein the mutant probes have a nucleotide sequence corresponding to one or more of SEQ ID NOs: 2, 6, 9, 12, 15, or 18, 21, 24, 27, 30, or 33.
80. A device for in-situ isothermal amplification and ligation, the device comprising:
The above detailed description of embodiments of the technology are not intended to be exhaustive or to limit the technology to the precise forms disclosed above. Although specific embodiments of, and examples for, the technology are described above for illustrative purposes, various equivalent modifications are possible within the scope of the technology as those skilled in the relevant art will recognize. For example, although steps are presented in a given order, alternative embodiments may perform steps in a different order. The various embodiments described herein may also be combined to provide further embodiments.
From the foregoing, it will be appreciated that specific embodiments of the technology have been described herein for purposes of illustration, but well-known structures and functions have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments of the technology. Where the context permits, singular or plural terms may also include the plural or singular term, respectively.
Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise,” “comprising,” and the like are to be construed in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to.” As used herein, the terms “connected,” “coupled,” or any variant thereof, means any connection or coupling, either direct or indirect, between two or more elements; the coupling of connection between the elements can be physical, logical, or a combination thereof. Additionally, the words “herein.” “above.” “below,” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of this application. Where the context permits, words in the above Detailed Description using the singular or plural number may also include the plural or singular number respectively. As used herein, the phrase “and/or” as in “A and/or B” refers to A alone, B alone, and A and B. Additionally, the term “comprising” is used throughout to mean including at least the recited feature(s) such that any greater number of the same feature and/or additional types of other features are not precluded. It will also be appreciated that specific embodiments have been described herein for purposes of illustration, but that various modifications may be made without deviating from the technology. Further, while advantages associated with some embodiments of the technology have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the technology. Accordingly, the disclosure and associated technology can encompass other embodiments not expressly shown or described herein.
The present application claims priority to U.S. Provisional Patent Application No. 62/672,882, titled “SYSTEM AND METHOD FOR LIGATION,” filed May 17, 2018, the disclosure of which is hereby incorporated by reference in its entirety.
This invention was made with government support under Grant Nos. AI027757 and R01 A1110375, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/033005 | 5/17/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62672882 | May 2018 | US |
