Methods and compositions according to the present disclosure relate generally to detection of analytes. According to specific aspects methods and compositions according to the present disclosure relate to detection of nucleic acid analytes using nanopore detection devices.
Specific and sensitive methods for detection of analytes is lacking in numerous fields. Nucleic acid detection methods are crucial for many applications, such as pathogen detection and genotyping. Many bio-sensing applications used fluorescent, bioluminescent, or colorimetric reporters for readouts, which often require optical sensing and additional design and synthesis of reporter molecules like fluorescence/quencher beacons or gold nanoparticles, implicating high cost. There is a continuing need for methods and devices for sensitive and specific detection of analytes.
Methods of detecting an analyte in a solution are provided according to aspects of the present disclosure which include: providing a nanopore counting device comprising; a first chamber and a second chamber; a barrier having a nanopore opening defined therein, the barrier separating the first chamber from the second chamber; a control/sensing system operable to apply an electrical potential between a solution in the first chamber and a solution in the second chamber and to sense a voltage and/or current between the chambers; disposing an ion-containing solution in the first and second chambers; disposing a sample in the first chamber, the sample containing or suspected of containing the analyte; applying an electrical potential between the solution in the first chamber and the solution in the second chamber; and sensing a baseline current between the chambers and detecting resistive pulses, wherein detecting resistive pulses provides a signal representative of presence of the analyte in the sample, and wherein no calibration step is performed before disposing the sample.
Methods of detecting an analyte in a solution using a nanopore counting device are provided according to aspects of the present disclosure, wherein the nanopore counting device includes a first chamber and a second chamber; a barrier having a nanopore opening defined therein, the barrier separating the first chamber from the second chamber; and a control/sensing system operable to apply an electrical potential between a solution in the first chamber and a solution in the second chamber and to sense a voltage and/or current between the chambers; the method including disposing an ion-containing solution in the first and second chambers; disposing a sample in the first chamber, the sample containing or suspected of containing the analyte; applying an electrical potential between the solution in the first chamber and the solution in the second chamber; and sensing a baseline current between the chambers and detecting resistive pulses, wherein detecting resistive pulses provides a signal representative of presence of the analyte in the sample, and wherein no calibration step is performed before disposing the sample.
According to aspects of the present disclosure, methods of detecting an analyte in a solution are provided wherein sensing a baseline current between the chambers and detecting resistive pulses provides a count of a number of molecules of analyte that pass through the nanopore during a period of time.
According to aspects of the present disclosure, methods of detecting an analyte in a solution, further include determining an estimated concentration of the molecules of analyte in the first chamber, wherein determining comprises calculation using the formula:
where:
Λ is the molar conductivity of the buffer solution in Siemens per meter per mole;
R is the translocation rate in resistive pulses per second;
μ is the free solution electrophoretic mobility of the analyte in meters per volt second;
NA is the Avogadro constant; and
Ib is the baseline current in nA.
According to aspects of the present disclosure, the barrier with the nanopore is a solid state nanopore barrier.
According to aspects of the present disclosure, methods of detecting an analyte in a solution are provided wherein the nanopore has an opening size larger than a size of an analyte molecule, the nanopore opening size being within one order of magnitude of the size of the analyte molecule.
According to aspects of the present disclosure, methods of detecting an analyte in a solution are provided wherein the period of time is determined by the number of resistive pulse, the number of resistive pulse during the period of time being at least 100.
According to aspects of the present disclosure, methods of detecting an analyte in a solution are provided wherein the number of resistive pulses is at least 200.
According to aspects of the present disclosure, methods of detecting an analyte in a solution are provided wherein the number of resistive pulses during the period of time is at least 200.
Methods of detecting an analyte in a solution are provided according to aspects of the present disclosure which include: providing a nanopore counting device comprising; a first chamber and a second chamber; a barrier having a nanopore opening defined therein, the barrier separating the first chamber from the second chamber; a control/sensing system operable to apply an electrical potential between a solution in the first chamber and a solution in the second chamber and to sense a voltage and/or current between the chambers; disposing an ion-containing solution in the first and second chambers; disposing a sample in the first chamber, the sample containing or suspected of containing the analyte; applying an electrical potential between the solution in the first chamber and the solution in the second chamber; and sensing a baseline current between the chambers and detecting resistive pulses, wherein detecting resistive pulses provides a signal representative of presence of the analyte in the sample, wherein no calibration step is performed before disposing the sample, and wherein the analyte is a target nucleic acid sequence.
Methods of detecting a nucleic acid analyte in a solution using a nanopore counting device are provided according to aspects of the present disclosure, wherein the nanopore counting device includes a first chamber and a second chamber; a barrier having a nanopore opening defined therein, the barrier separating the first chamber from the second chamber; and a control/sensing system operable to apply an electrical potential between a solution in the first chamber and a solution in the second chamber and to sense a voltage and/or current between the chambers; the method including disposing an ion-containing solution in the first and second chambers; disposing a sample in the first chamber, the sample containing or suspected of containing the analyte; applying an electrical potential between the solution in the first chamber and the solution in the second chamber; and sensing a baseline current between the chambers and detecting resistive pulses, wherein detecting resistive pulses provides a signal representative of presence of the analyte in the sample, and wherein no calibration step is performed before disposing the sample.
In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure a reporter nucleic acid is provided; a CRISPR-Cas system guide RNA (also known as a crRNA) that hybridizes to the target nucleic acid sequence is provided; and a CRISPR enzyme is provided, wherein the CRISPR enzyme is a Cas enzyme capable of forming a non-activated ribonucleoprotein (RNP) complex (non-activated RNP) with the guide RNA (also known as a crRNA), and wherein the non-activated RNP is capable of binding to the target nucleic acid sequence, thereby forming an activated RNP complex (activated RNP) having “trans” activity to indiscriminately cleave the reporter nucleic acid, wherein cleavage of the reporter nucleic acid results in at least two smaller cleaved portions of the reporter nucleic acid.
In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the crRNA (also known as a guide RNA) and Cas enzyme are contacted in a reaction vessel, thereby forming a non-activated complex of the crRNA and Cas enzyme, a non-activated RNP. The non-activated RNP is contacted with the sample containing or suspected of containing the target nucleic acid sequence in the same or in a different reaction vessel, and the target nucleic acid sequence and non-activated RNP specifically bind to each other if the target nucleic acid is present in the sample, thereby forming an activated RNP. The reporter nucleic acid is disposed in a reaction vessel with the activated RNP, wherein the activated RNP indiscriminately cleaves the reporter nucleic acid, wherein cleavage of the reporter nucleic acid results in at least two smaller cleaved portions of the reporter nucleic acid, and wherein cleavage of the reporter nucleic acid reduces passage of intact, non-cleaved reporter nucleic acid through the nanopore in the nanopore counting device such that a reduction of resistive pulses is produced which provides a signal representative of presence of the target nucleic acid sequence in the sample. A reaction vessel can be the first chamber of the nanopore counting device according to aspects of the present disclosure.
In methods of detecting a target DNA sequence analyte according to aspects of the present disclosure, the crRNA and a Cas12 enzyme are contacted in a reaction vessel, thereby forming a non-activated complex of the crRNA and Cas12 enzyme, a non-activated RNP. The non-activated RNP is contacted with the sample containing or suspected of containing the target DNA sequence in the same or in a different reaction vessel, and the target DNA sequence and non-activated RNP specifically bind to each other if the target DNA is present in the sample, thereby forming an activated RNP. The reporter nucleic acid is disposed in a reaction vessel with the activated RNP, wherein the activated RNP indiscriminately cleaves the reporter nucleic acid, wherein cleavage of the reporter nucleic acid results in at least two smaller cleaved portions of the reporter nucleic acid, and wherein cleavage of the reporter nucleic acid reduces passage of intact, non-cleaved reporter nucleic acid through the nanopore in the nanopore counting device such that a reduction of resistive pulses is produced which provides a signal representative of presence of the target DNA sequence in the sample. A reaction vessel can be the first chamber of the nanopore counting device according to aspects of the present disclosure.
In methods of detecting a target RNA sequence analyte according to aspects of the present disclosure, the crRNA and a Cas13 enzyme are contacted in a reaction vessel, thereby forming a non-activated complex of the crRNA and Cas13 enzyme, a non-activated RNP. The non-activated RNP is contacted with the sample containing or suspected of containing the target RNA sequence in the same or in a different reaction vessel, and the target DNA sequence and non-activated RNP specifically bind to each other if the target RNA is present in the sample, thereby forming an activated RNP. The reporter nucleic acid is disposed in a reaction vessel with the activated RNP, wherein the activated RNP indiscriminately cleaves the reporter nucleic acid, wherein cleavage of the reporter nucleic acid results in at least two smaller cleaved portions of the reporter nucleic acid, and wherein cleavage of the reporter nucleic acid reduces passage of intact, non-cleaved reporter nucleic acid through the nanopore in the nanopore counting device such that a reduction of resistive pulses is produced which provides a signal representative of presence of the target RNA sequence in the sample. A reaction vessel can be the first chamber of the nanopore counting device according to aspects of the present disclosure.
In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the reporter nucleic acid is a linear or circular single-stranded DNA molecule.
In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the reporter nucleic acid does not include a label, such as a fluorescent, radioactive or colored label.
In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the target nucleic acid sequence is a nucleic acid of a microorganism.
In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the target nucleic acid sequence is a nucleic acid of a virus, a bacterium, a fungus, or a parasite.
In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the sample is obtained from a mammal. In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the sample is derived from a human.
In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the sample contains one or more amplified nucleic acids. In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the sample is protein-free or substantially protein-free.
In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the sample is derived from a mammal having, or suspected of having, an infection by a virus, a bacterium, a fungus, or a parasite.
In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the sample is derived from a human having, or suspected of having, an infection by a virus, a bacterium, a fungus, or a parasite.
In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the sample is an environmental sample, containing, or suspected of containing, a virus, a bacterium, a fungus, or a parasite.
In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the target nucleic acid sequence is a nucleic acid of a human immunodeficiency virus.
In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the target nucleic acid sequence is a nucleic acid of a coronavirus.
In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the target nucleic acid sequence is a SARS-Cov-2 coronavirus nucleic acid sequence.
In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the sample is derived from a plant.
In methods of detecting a target nucleic acid sequence analyte according to aspects of the present disclosure, the sample is derived from a plant having, or suspected of having, an infection by a virus, a bacterium, a fungus, or a parasite.
Methods of detecting a target nucleic acid sequence in a solution are provided according to aspects of the present disclosure which include: providing a nanopore counting device comprising; a first chamber and a second chamber; a barrier having a nanopore opening defined therein, the barrier separating the first chamber from the second chamber; a control/sensing system operable to apply an electrical potential between a solution in the first chamber and a solution in the second chamber and to sense a voltage and/or current between the chambers; calibrating the nanopore counting device to determine a rate of translocation of molecules of a calibrant from a calibration solution through the nanopore when a calibrating electrical potential is applied between the chambers; disposing an ion-containing solution in the first and second chambers; providing a reporter nucleic acid; providing a CRISPR-Cas system guide RNA (crRNA) that specifically hybridizes to the target nucleic acid sequence; providing a CRISPR enzyme, wherein the CRISPR enzyme is a Cas enzyme capable of forming a non-activated ribonucleoprotein (RNP) complex (non-activated RNP) with the guide RNA, and wherein the non-activated RNP is capable of binding to the target nucleic acid sequence, forming an activated RNP complex (activated RNP) having “trans” activity to cleave the reporter nucleic acid; contacting the crRNA and Cas enzyme, thereby forming the non-activated RNP; disposing the non-activated RNP in the first chamber with the sample, wherein the non-activated RNP and the target nucleic acid sequence specifically bind if the target nucleic acid is present in the sample, forming an activated, wherein the activated cleaves the reporter nucleic acid; applying an electrical potential between the solution in the first chamber and the solution in the second chamber; and sensing current between the first chamber and the second chamber and detecting resistive pulses, wherein cleavage of the reporter nucleic acid reduces passage of the reporter nucleic acid through the nanopore such that a reduction of resistive pulses is produced which provides a signal representative of presence of the target nucleic acid sequence in the sample. According to aspects of the present disclosure, the barrier with the nanopore barrier is a solid state nanopore barrier or a biological nanopore barrier.
According to aspects of the present disclosure, the step of detecting resistive pulses further comprises counting resistive pulses to determine a number of reporter nucleic acid molecules that pass through the nanopore during a period of time, thereby determining a rate of translocation of the reporter nucleic acid molecules.
According to aspects of the present disclosure, methods of detecting a target nucleic acid sequence analyte further include determining the estimated concentration of the target nucleic acid sequence in the first chamber based on the reporter nucleic acid translocation rate as compared to the calibrant translocation rate.
According to aspects of the present disclosure, the calibrating step comprises: disposing an ion-containing solution in the first and second chamber and a known concentration of calibrant molecules in the first chamber, the calibrant molecules being the same or similar to the reporter nucleic acid molecules; applying the calibrating electrical potential between the chambers; sensing current between the chambers and counting resistive pulses to determine a number of molecules of the calibrant that pass through the nanopore during a period of time; and determining a rate of translocation for the known concentration of calibrant at the calibrating electrical potential.
According to aspects of the present disclosure, the calibrant molecules are the same as the reporter nucleic acid molecules, the calibrating electrical potential is in the range of 0.5 to 2 times the electrical potential used after the calibrating step, and the ion-containing solution during is the same during the calibrating step and after the calibrating step.
According to aspects of the present disclosure, the target nucleic acid sequence is DNA and the Cas enzyme is a Cas12 enzyme. According to aspects of the present disclosure, the target nucleic acid sequence is RNA and the Cas enzyme is a Cas13 enzyme. According to aspects of the present disclosure, the reporter nucleic acid is a linear circular single-stranded DNA molecule. According to aspects of the present disclosure, the reporter nucleic acid does not include a label. According to aspects of the present disclosure, the target nucleic acid sequence is a nucleic acid of a microorganism. According to aspects of the present disclosure, the target nucleic acid sequence is a nucleic acid of a virus, a bacterium, a fungus, or a parasite. According to aspects of the present disclosure, the sample is obtained from a mammal. According to aspects of the present disclosure, the sample is derived from a human. According to aspects of the present disclosure, the sample is derived from a mammal having, or suspected of having, an infection by a virus, a bacterium, a fungus, or a parasite. According to aspects of the present disclosure, the sample is derived from a human having, or suspected of having, an infection by a virus, a bacterium, a fungus, or a parasite.
According to aspects of the present disclosure, the sample is an environmental sample, containing, or suspected of containing, a virus, a bacterium, a fungus, or a parasite.
According to aspects of the present disclosure, the target nucleic acid sequence is a nucleic acid of a human immunodeficiency virus.
According to aspects of the present disclosure, the target nucleic acid sequence is a nucleic acid of a coronavirus. According to aspects of the present disclosure, the coronavirus is a SARS-Cov-2 coronavirus. According to aspects of the present disclosure, the sample is derived from a plant.
According to aspects of the present disclosure, the sample is derived from a plant having, or suspected of having, an infection by a virus, a bacterium, a fungus, or a parasite.
Scientific and technical terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. Such terms are found defined and used in context in various standard references illustratively including M. Green and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th Ed., 2012; F. M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002; D. Clark et al., Molecular Biology, 3rd Ed., Academic Cell, 2018; CRISPR/Cas: A Laboratory Manual, Doudna and Mali (eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA, 2016; D. L. Nelson and M. M. Cox, Lehninger Principles of Biochemistry, 4th Ed., W.H. Freeman & Company, 2004; and Herdewijn, P. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004.
The singular terms “a,” “an,” and “the” are not intended to be limiting and include plural referents unless explicitly stated otherwise or the context clearly indicates otherwise.
The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/−10% or less, +/−5% or less, +/−1% or less, and +/−0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.
Reference herein to “one aspect”, “an aspect,” “an example,” means that a particular feature, structure or characteristic described or named is included in at least one embodiment of the present invention. Thus, appearances of the phrases “according to aspects,” “according to an aspect,” or “an example” herein are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination
The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.
Nanopore Digital Counting of Single Molecules
According to an aspect of the present invention, a concentration of molecules of an analyte in a solution may be determined using a nanopore digital counting method.
According to an aspect of the present invention, nanopore devices are not modified to include specific binding sites for the analyte. Thus, the nanopore device may be used to detect any of various analytes, including nucleic acid analytes.
Nanopore-based sensors allow single molecules to be analyzed electronically without the need for labeling and partitioning, unlike with optical digital counting or bulk analog sensing. Conceptually, a nanopore sensor represents an ideal single molecule counting device due to its unique features of label-free electronic sensing, single-molecule sensitivity, and potential reusability.
The barrier may take a variety of forms, including biological barriers with a biological nanopore and solid state nanopores where the nanopore is created using any one of a variety of techniques.
Molecules of an analyte are represented as small spheres at 26 and are illustrated as present in both of the chambers. As will be clear to those of skill in the art, the drawing is not to scale. In one example, where the molecules of analyte 26 are DNA, the molecules have a width dimension of approximately 2 nm (nanometers) and the nanopore has a width dimension of approximately 10 nm. The nanopore is typically larger than the molecules to be analyzed, such as up to one order of magnitude larger, though other dimensions may be used.
In the presence of an electrical potential, provided by the electrodes 22 and 24, molecules of analyte translocate through the nanopore from one chamber to the other electrophoretically. When a molecule 26 is not translocating through the nanopore, current will flow through the solution at a baseline rate. When a molecule translocates, the current flow is partially obstructed causing resistance to current flow to rise and current flow to drop.
By counting the number of molecules that translocate through the nanopore during a period of time, a translocation rate may be determined. The translocation rate depends on the concentration of the molecules of analyte in the first chamber, the strength of the electrical potential, the size and the shape of the nanopore and various other factors. The strength of the electrical potential is controlled by the user and the size of the nanopore and most other factors are generally stable over time, so the rate of translocation therefore correlates with the analyte concentration. Put another way, a single molecule is electrophoretically driven through the nanopore, a detectable ionic current blockade generates a digital “1” signal, the rate of which is proportional to the sample concentration. Resolving this digital event itself is much easier than analyzing its analog features such as magnitude and duration of the current dip.
Existing theories and experiments have shown that when interactions between molecules are negligible, the molecule molar concentration (mol/m3) is linearly related to the translocation rate (events per second) as R=αNAC, where NA is the Avogadro constant and α is usually referred to as the capture rate. Since capture rate α strongly depends on experimental parameters such as nanopore geometry, temperature, molecule size, and applied voltage, it is typically necessary to define a calibration curve of the rate versus concentration to then use the nanopore digital counting method to infer the concentration in an unknown sample. Moreover, the calibration curve must be obtained under the same experimental conditions such that there is a comparable capture rate α. Unfortunately, generating this calibration curve is often time-consuming and challenging due to nanopore clogging issues.
Calibration may be performed in several ways, as long as the calibration results in determining a translocation rate versus concentration slope under conditions sufficiently similar to the conditions to be used when testing a sample with an unknown concentration. Also, the calibration should be performed with a known concentration that is not too much different from the unknown concentration. For example, if the calibration is performed with a first concentration and the unknown concentration is several orders of magnitude greater, the slope determined during the calibration step may not be applicable. This could necessitate calibration at multiple concentrations or performing a new calibration.
One approach to calibration is as follows. Starting with the same nanopore counting device to be used in the test, an ion-containing solution is disposed in the first and second chamber and a known concentration of calibrant molecules is disposed in the first chamber. As noted, the calibrant molecules need to be functionally the same or very similar to what will be used in the actual test. Typically, the same analyte molecules are used. A calibrating electrical potential is applied between the chambers to cause translocation of calibrant molecules. The calibrating electrical potential should be the same as will be used during the test. In some cases, calibration may be performed at more than one level of electrical potential so that other levels may be used during testing. In some examples, the rate determined during calibration may be applicable to potentials that are someone larger or smaller, with appropriate adjustment. For example, in some cases, the calibrating electrical potential may be in the range of 0.1 to 10 times the electrical potential used in the actual test.
Current between the chambers is then sensed and resistive pulses are digitally counted to determine a number of molecules of the calibrant that pass through the nanopore during a period of time. The interval between translocation events varies, generally indicating a Poisson process, as will be discuss later. In order to reduce uncertainty in the rate determined during calibration, the rate should be determined for a statistically significant number of translocation events. This will be further discussed later. Once enough events have been counted, a rate of translocation for the known concentration of calibrant at the calibrating electrical potential. The testing of the unknown concentration may then proceed, with the concentration being determined by determining a rate and then dividing by the slope of the rate versus concentration curve from the calibration.
Calibration-Free Nanopore Digital Counting of Single Molecules
According to a further aspect of the present invention, a method is provided for determining a concentration of molecules of an analyte in a solution using nanopore digital counting without the need for a separate calibration step prior to testing the unknown solution.
The calibration-free method makes use of a nanopore counting device that may be the same as described above with reference to
An embodiment of the calibration-free method will be described, and then details of the theory and testing leading to the embodiment will be provided. A method of determining an estimated concentration of an analyte in a solution starts with providing a nanopore counting device.
The barrier may take a variety of forms, though typically solid state nanopores are used, where the nanopore is created using one of a variety of techniques. In an example that will be described below, a glass nanopore was used.
Molecules of an analyte are represented as small spheres at 26 and are illustrated as present in both of the chambers. As will be clear to those of skill in the art, the drawing is not to scale. In one example, where the molecules of analyte 26 are DNA, the molecules have a width dimension of approximately 2 nm (nanometers) and the nanopore has a width dimension of approximately 10 nm. The nanopore is typically larger than the molecules to be analyzed, such as up to one order of magnitude larger, though other dimensions may be used.
In the presence of an electrical potential, provided by the electrodes 22 and 24, molecules of analyte translocate through the nanopore from one chamber to the other. When a molecule 26 is not translocating through the nanopore, current will flow through the solution at a baseline rate. When a molecule translocates, the current flow is partially obstructed causing resistance to current flow to rise and current flow to drop.
By counting the number of molecules that translocate through the nanopore during a period of time, a translocation rate is determined.
Unlike the above-described method, the calibration-free method includes sensing a baseline current between the chambers in addition to counting resistive pulses to determine a number of molecules of analyte that pass through the nanopore during the period of time. The baseline current is the current when a molecule is not translocating. Put another way, the baseline current is the current between resistive pulses. As will be clear to those of skill in the art, the baseline current is typically not a constant value. Instead, the current signal typically fluctuates and may be noisy. As such, the baseline current may be an average or normalized current determined based on the fluctuating baseline current signal. The longer that baseline current is sensed, the better the average will be.
As with the earlier method, translocation events should be counted for long enough to reduce the uncertainty to an acceptable level. Once a translocation rate and a baseline current are determined, the estimated concentration of the molecules of analyte in the first chamber may be determined using the following formula:
where:
As noted previously, no calibration step is performed before the step of disposing the solution with the unknown concentration in the first chamber.
The development of the calibration-free method will now be described. The inventors first studied the statistics of the molecule translocation rate and developed an experimentally practical method to measure the rate. They then developed a quantitative model for molecule quantification without the need for prior knowledge of experimental conditions such as nanopore geometry, size, and applied voltage. This was achieved by using the background ions as the in situ reference such that the molecule translocation rates were normalized to the ion translocation rates (baseline current). This model was experimentally validated for different nanopores and DNA molecules with different sizes. While the results discussed below were from glass nanopores and DNA molecules, the principle could be well extended to other nanopore types and other charged molecules.
Glass nanopore characterization was performed by standard I-V (current-voltage) measurement, SEM, and TEM imaging. For I-V characterization, the glass nanopore was filled with 1 M KCl in a Tris-EDTA buffer by a microinjector and then immersed in the testing solution. Ag/AgCl electrodes 52 and 54 were used for interfacing the electrical measurement, and the I-V curve was recorded for nanopore size estimation, as shown in
The schematic of the single molecule counting setup is illustrated in
It was previously observed that the mean time between single-molecule capture events in a solid-state nanopore follows an exponential distribution, indicating a Poisson process. To validate if this was also true in the glass nanopore, studies were performed on λ-DNAs with a serial of concentrations ranging from 12 to 60 pM. A quick eyeball on the current time traces in
With an experimentally efficient n/T approach to determine the rate, the next task was to determine the capture rate α. The dynamics of molecule translocation through the nanopore consists of three steps: (1) the molecule moves from the bulk of the reaction chamber toward the pore entrance by a combination of diffusion and drift forces; (2) the molecule is captured at the entrance of the nanopore; and (3) the molecule overcomes an entropy energy barrier and goes through the nanopore, causing a detectable ionic current blockade which can be detected electronically as a digital signal. It is known that the capture rate α could be diffusion limited (step 1) or barrier limited (step 3). The glass nanopores used in the experiments are around 10 nm in size, which is large enough such that the transport is diffusion limited rather than barrier limited, as indicated by the linear dependence of the capture rate on the voltage.
The derivation of capture rate for the conical shaped nanopore is as follows. First, a length scale r* is introduced such that at the distances r>r* DNA is freely diffusing in the bulk solution, with potential V (r) playing a marginal role, while at r<r*, DNA gets irreversibly captured and funneled down the potential V (r*) directly to the pore mouth. If r* is estimated, then the diffusion-controlled rate is given by the classical Smoluchowski result, α=2πDr* where D is the diffusion coefficient. Assuming that the equipotential surfaces are semi-spherical outside the pore, one obtains the electrostatic potential V(r)=I/2πσr where σ is the conductivity of the nanopore and current I can be estimated by I=GΔV. For a conical nanopore, conductance can be expressed as G=σ[4l/πdtdb+1/2dt+1/2db]−1 where l, dt, and db are the length and diameters of the truncated cone. Hence by defining the characteristic length d as d=1/2π[4l/πdtdb+1/2dt+1/2db]−1, we have V(r)=ΔVd/r. Based on the previous studies, it is knows that V(r*)=D/μ, where μ is the free solution electrophoretic mobility. So r* can be estimated by r*=μd/D*ΔV. Considering the equation α=2πDr*, from above, we can derive the capture rate for a conical shape nanopore as α=2πμd ΔV.
In the diffusion-limited region, the capture rate for the conical-shaped glass nanopore is given by α=2πμdΔV, where p is the free solution electrophoretic mobility, ΔV is the applied electric potential across the pore, and d is the characteristic length of the nanopore. If the nanopore geometry and size is explicitly known for a particular experiment, the capture rate can be directly calculated to determine the unknown sample concentration without calibration, similar to a pressure-driven calibration-less quantitation of nanoparticles by calculating the hydrodynamic resistance. Nevertheless, it is well-known that glass nanopore geometry is widely dispersed. TEM characterization of each nanopore is often destructive and is time-, facility-, and expertise-intensive. In addition, experimental conditions such as applied voltage, temperatures, and buffers also vary from one experiment to the other. To properly determine the unknown sample concentration, a calibration curve must be obtained under the same experimental conditions to extract the capture rate α in that particular experiment. While this could be done, it is often time-consuming and experimentally challenging due to potential nanopore clogging under repetitive testing.
To overcome these challenges, an aspect of the present invention was developed, providing an in situ method for determining the capture rate α without the need for prior knowledge of nanopore experimental conditions. This is achieved by recognizing that the baseline current carries information about the background ion translocation rate, as shown in
The baseline current can be estimated as follows.
The baseline current is estimated based on the applied voltage ΔV and the nanopore conductance by
I
b
=GΔV
For a conical nanopore, conductance can be expressed as
Similar to the last case, by defining the characteristic length
we have:
G=2πσd
The conductivity as a function of ion species concentration and ion mobility can be written as:
where NA is the Avogadro constant, zi is the valance, Ci is the molar concentration [mole/m3], and e represents the elementary charge (1.6×10-19C), μi is the ions mobility [m2/(V·s)]. Assuming monovalent ions, we can write Ci=Cion. As a result,
Defining the molar conductivity as
The baseline current can thus be written and estimated as Ib=2πΛCiondΔV, where A is the molar conductivity which depends on the mobility and valence of the ions as Λ=ΣiNAeziμi. The previously inaccessible parameter α=2πμdΔV can be rewritten as
This equation implies that the unknown capture rate can be derived from the experimentally accessible baseline current and the ionic concentration without knowing the nanopore geometry, size, and the applied voltage. The molecule mobility μ and molar conductivity Λ can be estimated for a particular molecule and salt. Thus, the molecule translocation rate R=αNACmol can be written as
To validate this equation, experiments were performed with 10 kbp DNA at 24 pM in the 1 M KCl buffer solution.
This equation shows that an unknown sample concentration can be quantified without explicitly knowing the nanopore geometry, size, and the applied voltage, as long as the parameters on the right-hand side of the equation could be determined. To validate this method, tests were performed with λ-DNA, 5 kbp DNA, and 10 kbp DNA at five known concentrations (12, 24, 36, 48, and 60 pM) in 1 M KCl buffer, intentionally using glass nanopores pulled from different batches. Since the free solution electrophoretic mobility of DNA in the Tris-EDTA buffer was theoretically and experimentally shown to be independent of the DNA length longer than a few persistence lengths, μ of 4.5×10−8 m2 V−1 s−1 was used for all DNA molecules. The buffer solution is dominated by 1 M KCl, and thus the molar conductivity A is estimated to be 10.86 m−1 M−1 S. Table 1 summarizes the results for this calibration-free method for concentration measurement.
The baseline current (Ib) and translocation rate (R) was determined from the experiment.
Nucleic Acid Assays
According to aspects of the present disclosure, the analyte is a target nucleic acid sequence. The target nucleic acid sequence is RNA or DNA.
The term “nucleic acid” as used herein refers to RNA or DNA molecules having more than one nucleotide in any form including single-stranded, double-stranded, oligonucleotide or polynucleotide. The terms “nucleotide sequence” and “nucleic acid sequence” refer to the ordering of nucleotides in an oligonucleotide or polynucleotide in reference to a single-stranded form of nucleic acid.
The term “nucleic acid” further encompasses any chemical modifications of RNA or DNA molecules, such as inclusion of one or more modified or non-naturally occurring nucleotides. Such modified or non-naturally occurring nucleotides include, but are not limited to, 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, methylations, and non-standard base-pairing combinations, such as isobases, such as deoxyisocytidine and deoxyisoguanosine. Accordingly, the nucleic acids described herein include not only the standard bases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) but also non-standard or non-naturally occurring nucleotides.
The term “double-stranded” is used herein to refer to nucleic acids characterized by binding interaction of complementary nucleotide sequences. A double-stranded nucleic acid includes a “sense” strand and an “antisense” strand. Such duplexes include RNA/RNA, DNA/DNA or RNA/DNA types of duplexes.
The term “single-stranded” is used to refer to nucleic acids not bound by binding interaction to a complementary nucleotide sequence.
According to aspects of the present disclosure, a sample obtained from a subject or an environmental sample is assayed for a target nucleic acid according to aspects of the present disclosure.
The subject can be any organism including, a mammalian organism, a vertebrate non-mammalian organism, or a plant.
A mammalian subject can be any mammal including, but not limited to, a human; a non-human primate; a rodent such as a mouse, rat, or guinea pig; a domesticated pet such as a cat or dog; a horse, cow, pig, deer, sheep, goat, or rabbit. A non-mammalian vertebrate subject can be any vertebrate organism including, but not limited to, a bird such as a duck, goose, chicken, or turkey, a reptile, or an amphibian. Subjects can be either gender and can be any age. In aspects of methods of detecting a target nucleic acid sequence, the subject is human.
According to aspects of the present disclosure, the subject is an individual infected, or suspected of being infected, by a microorganism.
The term “sample” or “biological sample” as used herein refers to material obtained from any suitable source, including, a subject or an environment.
According to aspects of the present disclosure, a sample obtained from a subject can be any material containing, or suspected of containing, the target nucleic acid. Exemplary samples include, but are not limited to, a cell sample, a tissue sample, a fluid sample or a combination of two or more thereof. Exemplary samples include, but are not limited to, whole blood, serum, plasma, blood cells, lymph, bronchoalveolar lavage material, cerebrospinal fluid, mucus, saliva, semen, sweat, tears, amniotic fluid, wound material such as pus or a wound exudate, skin, biopsy material, synovial fluid, gastrointestinal material, vaginal fluid, fecal material, sputum, urine, any other body fluid, cell, tissue, or any material obtained from a subject that contains, or is suspected of containing a target nucleic acid, or a combination of two or more thereof.
According to aspects of the present disclosure, a sample obtained from a plant can be any portion of a plant containing, or suspected of containing, the target nucleic acid. Exemplary plant samples include, but are not limited to, a cell sample, a tissue sample, a fluid sample or a combination of two or more thereof.
Sample collection procedures for obtaining a sample from a subject are known in the art are suitable for use with various aspects of the present disclosure.
According to aspects of the present disclosure, a sample obtained from an environment can be any material containing, or suspected of containing, the target nucleic acid. Exemplary environmental samples include, but are not limited to, soil samples, air samples, water samples, aerosol samples, or a combination of any two or more thereof. An environmental sample may be, without limitation, obtained from an area, object, space, material, or a combination of any two or more thereof, such as a swab, scrape, wipe, or portion of a hospital room surface or object, clothing, furniture, equipment, food, drink, or any other object, surface, or material.
According to aspects of the present disclosure, a sample is purified to enrich for a target nucleic acid. The term “purified” in the context of a sample refers to separation of a target nucleic acid in the sample, or suspected of being present in the sample, from at least one other component present in the sample.
According to aspects of the present disclosure, a sample is purified to enrich for a target nucleic acid and remove, or substantially remove, proteins from the sample. According to aspects of the present disclosure, a sample is protein-free, or substantially protein-free, such as containing no detectable protein, less than 1 nM protein concentration, or less than 1 μM protein concentration. Detection of proteins and/or quantitation of proteins in a sample can be accomplished by any of various protein detection and/or quantitation assays, such as, but not limited to, Bradford or Lowry methods.
The term “sample” also include samples processed to enrich for nucleic acids such as by decrease or removal of other non-nucleic acid components of the sample, and/or by amplification of nucleic acids in the sample, such as by amplification of a target nucleic acid in the sample.
Amplification of a target nucleic acid is achieved using an in vitro amplification method. The term “amplification” refers to copying a target nucleic acid, thereby producing copies of the target nucleic acid.
The term “amplification” refers to methods which include template directed primer extension catalyzed by a nucleic acid polymerase using at least one primer, and in particular methods, a pair of primers which flank the target nucleic acid. Such methods include, but are not limited to, Polymerase Chain Reaction (PCR), reverse-transcription PCR (RT-PCR). ligation-mediated PCR (LM-PCR), phi-29 PCR, and other nucleic acid amplification methods, for instance, as described in C. W. Dieffenbach et al., PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2003; and V. Demidov et al., DNA Amplification: Current Technologies and Applications, Taylor & Francis, 2004.
In particular embodiments, nucleic acids are optionally substantially purified from the sample to produce a substantially purified nucleic acid sample for use in an inventive assay. The term “substantially purified” refers to a desired material separated from other substances naturally present in a sample obtained from the subject so that the desired material makes up at least about 0.01-100% of the mass, by weight, such as about 0.01%, 0.1%, 1%, 5%, 10%, 25%, 50% 75% or greater than about 75% of the mass, by weight, of the substantially purified sample. Purification is achieved by techniques illustratively including electrophoretic methods such as gel electrophoresis and 2-D gel electrophoresis; solvent-based removal of proteins; and precipitation.
According to aspects of the present disclosure, the target nucleic acid sequence is a nucleic acid of a microorganism present in a sample obtained from a subject. The microorganism to be detected in a sample via detection of a target nucleic acid sequence of the microorganism can be any microorganism, such as, but not limited to bacteria, viruses, fungi, and parasite microorganisms, such as, but not limited to, protozoa.
Methods according to aspects of the present disclosure can be used to detect various types of bacteria, including pathogens and bacteria which are not ordinarily pathogenic (e.g. normal microflora of the gut) including, but not limited to bacteria of any of the following genera: Acidilobus, Aeropyrum, Archaeoglobus, Caldisphaera, Caldivirga, Desulfirococcus, Desulfurolobus, Ferroglobus, Ferroplasma, Geoglobus, Haloarcula, Halobacterium, Halobaculum, Halobiforma, Halococcus, Haloferax, Halogeometricum, Halomethanococcus, Halorhabdus, Halorubrobacterium, Halorubrum, Halosimplex, Haloterrigena, Hyperthermus, Ignicoccus, Metallosphaera, Methanimicrococcus, Methanobacterium, Methanobrevibacter, Methanocalculus, Methanocaldococcus, Methanococcoides, Methanococcus, Methanocorpusculum, Methanoculleus, Methanofollis, Methanogenium, Methanohalobium, Methanohalophilus, Methanolacinia, Methanolobus, Methanomicrobium, Methanomicrococcus, Methanoplanus, Methanopyrus, Methanosaeta, Methanosalsum, Methanosarcina, Methanosphaera, Methanospirillum, Methanothermobacter, Methanothermococcus, Methanothermus, Methanothrix, Methanotorris, Natrialba, Natrinema, Natronobacterium, Natronococcus, Natronomonas, Natronorubrum, Palaeococcus, Picrophilus, Pyrobaculum, Pyrococcus, Pyrodictium, Pyrolobus, Staphylothermus, Stetteria, Stygiolobus, Sulfolobus, Sulfophobococcus, Sulfurisphaera, Sulfurococcus, Thermocladium, Thermococcus, Thermodiscus, Thermofilum, Thermoplasma, Thermoproteus, Thermosphaera, and Vulcanisaeta.
Methods according to aspects of the present disclosure can be used to detect various types of bacteria, including pathogens and bacteria which are not ordinarily pathogenic (e.g. normal microflora of the gut) including, but not limited to bacteria of any of the following genera: Abiotrophia, Acetitomaculum, Acetivibrio, Acetoanaerobium, Acetobacter, Acetobacterium, Acetofilamentum, Acetogenium, Acetohalobium, Acetomicrobium, Acetonema, Acetothermus, Acholeplasma, Achromatium, Achromobacter, Acidaminobacter, Acidaminococcus, Acidimicrobium, Acidiphilium, Acidisphaera, Acidithiobacillus, Acidobacterium, Acidocella, Acidomonas, Acidothermus, Acidovorax, Acinetobacter, Acrocarpospora, Actinoalloteichus, Actinobacillus, Actinobaculum, Actinobispora, Actinocorallia, Actinokineospora, Actinomadura, Actinomyces, Actinoplanes, Actinopolymorpha, Actinopolyspora, Actinopycnidium, Actinosporangium, Actinosynnema, Aegyptianella, Aequorivita, Aerococcus, Aeromicrobium, Aeromonas, Afipia, Agitococcus, Agreia, Agrobacterium, Agrococcus, Agromonas, Agromyces, Ahrensia, Albibacter, Albidovulum, Alcaligenes, Alcalilimnicola, Alcanivorax, Algoriphagus, Alicycliphilus, Alicyclobacillus, Alishewanella, Alistipes, Alkalibacterium, Alkalilimnicola, Alkaliphilus, Alkalispirillum, Alkanindiges, Allisonella, Allochromatium, Allofustis, Alloiococcus, Allomonas, Allorhizobium, Alterococcus, Alteromonas, Alysiella, Amaricoccus, Aminobacter, Aminobacterium, Aminomonas, Ammonifex, Ammoniphilus, Amoebobacter, Amorphosporangium, Amphibacillus, Ampullariella, Amycolata, Amycolatopsis, Anaeroarcus, Anaerobacter, Anaerobaculum, Anaerobiospirillum, Anaerobranca, Anaerococcus, Anaerofilum, Anaeroglobus, Anaerolinea, Anaeromusa, Anaeromyxobacter, Anaerophaga, Anaeroplasma, Anaerorhabdus, Anaerosinus, Anaerostipes, Anaerovibrio, Anaerovorax, Anaplasma, Ancalochloris, Ancalomicrobium, Ancylobacter, Aneurinibacillus, Angiococcus, Angulomicrobium, Anoxybacillus, Anoxynatronum, Antarctobacter, Aquabacter, Aquabacterium, Aquamicrobium, Aquaspirillum, Aquifex, Arachnia, Arcanobacterium, Archangium, Arcobacter, Arenibacter, Arhodomonas, Arsenophonus, Arthrobacter, Asaia, Asanoa, Asteroleplasma, Asticcacaulis, Atopobacter, Atopobium, Aurantimonas, Aureobacterium, Azoarcus, Azomonas, Azomonotrichon, Azonexus, Azorhizobium, Azorhizophilus, Azospira, Azospirillum, Azotobacter, Azovibrio, Bacillus, Bacterionema, Bacteriovorax, Bacteroides, Bactoderma, Balnearium, Balneatrix, Bartonella, Bdellovibrio, Beggiatoa, Beijerinckia, Beneckea, Bergeyella, Beutenbergia, Bifidobacterium, Bilophila, Blastobacter, Blastochloris, Blastococcus, Blastomonas, Blattabacterium, Bogoriella, Bordetella, Borrelia, Bosea, Brachybacterium, Brachymonas, Brachyspira, Brackiella, Bradyrhizobium, Branhamella, Brenneria, Brevibacillus, Brevibacterium, Brevinema, Brevundimonas, Brochothrix, Brucella, Brumimicrobium, Buchnera, Budvicia, Bulleidia, Burkholderia, Buttiauxella, Butyrivibrio, Caedibacter, Caenibacterium, Calderobacterium, Caldicellulosiruptor, Caldilinea, Caldimonas, Caldithrix, Caloramator, Caloranaerobacter, Calymmatobacterium, Caminibacter, Caminicella, Campylobacter, Capnocytophaga, Capsularis, Carbophilus, Carboxydibrachium, Carboxydobrachium, Carboxydocella, Carboxydothermus, Cardiobacterium, Camimonas, Carnobacterium, Caryophanon, Caseobacter, Catellatospora, Catenibacterium, Catenococcus, Catenuloplanes, Catonella, Caulobacter, Cedecea, Cellulomonas, Cellulophaga, Cellulosimicrobium, Cellvibrio, Centipeda, Cetobacterium, Chainia, Chelatobacter, Chelatococcus, Chitinophaga, Chlamydia, Chlamydophila, Chlorobaculum, Chlorobium, Chloroflexus, Chloroherpeton, Chloronema, Chondromyces, Chromatium, Chromobacterium, Chromohalobacter, Chryseobacterium, Chryseomonas, Chrysiogenes, Citricoccus, Citrobacter, Clavibacter, Clevelandina, Clostridium, Cobetia, Coenonia, Collinsella, Colwellia, Comamonas, Conexibacter, Conglomeromonas, Coprobacillus, Coprococcus, Coprothermobacter, Coriobacterium, Corynebacterium, Couchioplanes, Cowdria, Coxiella, Craurococcus, Crenothrix, Crinalium (not validly published), Cristispira, Croceibacter, Crocinitomix, Crossiella, Cryobacterium, Cryomorpha, Cryptobacterium, Cryptosporangium, Cupriavidus, Curtobacterium, Cyclobacterium, Cycloclasticus, Cystobacter, Cytophaga, Dactylosporangium, Dechloromonas, Dechlorosoma, Deferribacter, Defluvibacter, Dehalobacter, Dehalospirillum, Deinobacter, Deinococcus, Deleya, Delftia, Demetria, Dendrosporobacter, Denitrobacterium, Denitrovibrio, Dermabacter, Dermacoccus, Dermatophilus, Derxia, Desemzia, Desulfacinum, Desulfitobacterium, Desulfobacca, Desulfobacter, Desulfobacterium, Desulfobacula, Desulfobulbus, Desulfocapsa, Desulfocella, Desulfococcus, Desulfofaba, Desulfofrigus, Desulfofustis, Desulfohalobium, Desulfomicrobium, Desulfomonas, Desulfomonile, Desulfomusa, Desulfonatronovibrio, Desulfonatronum, Desulfonauticus, Desulfonema, Desulfonispora, Desulforegula, Desulforhabdus, Desulforhopalus, Desulfosarcina, Desulfospira, Desulfosporosinus, Desulfotalea, Desulfotignum, Desulfotomaculum, Desulfovibrio, Desulfovirga, Desulfurella, Desulfurobacterium, Desulfuromonas, Desulfuromusa, Dethiosulfovibrio, Devosia, Dialister, Diaphorobacter, Dichelobacter, Dichotomicrobium, Dictyoglomus, Dietzia, Diplocalyx, Dolosicoccus, Dolosigranulum, Dorea, Duganella, Dyadobacter, Dysgonomonas, Ectothiorhodospira, Edwardsiella, Eggerthella, Ehrlichia, Eikenella, Elytrosporangium, Empedobacter, Enhydrobacter, Enhygromyxa, Ensifer, Enterobacter, Enterococcus, Enterovibrio, Entomoplasma, Eperythrozoon, Eremococcus, Erwinia, Erysipelothrix, Erythrobacter, Erythromicrobium, Erythromonas, Escherichia, Eubacterium, Ewingella, Excellospora, Exiguobacterium, Facklamia, Faecalibacterium, Faenia, Falcivibrio, Ferribacterium, Ferrimonas, Fervidobacterium, Fibrobacter, Filibacter, Filifactor, Filobacillus, Filomicrobium, Finegoldia, Flammeovirga, Flavimonas, Flavobacterium, Flectobacillus, Flexibacter, Flexistipes, Flexithrix, Fluoribacter, Formivibrio, Francisella, Frankia, Frateuria, Friedmanniella, Frigoribacterium, Fulvimarina, Fulvimonas, Fundibacter, Fusibacter, Fusobacterium, Gallibacterium, Gallicola, Gallionella, Garciella, Gardnerella, Gelidibacter, Gelria, Gemella, Gemmata, Gemmatimonas, Gemmiger, Gemmobacter, Geobacillus, Geobacter, Geodermatophilus, Georgenia, Geothrix, Geotoga, Geovibrio, Glaciecola, Globicatella, Gluconacetobacter, Gluconoacetobacter, Gluconobacter, Glycomyces, Gordonia, Gordonia, Gracilibacillus, Grahamella, Granulicatella, Grimontia, Haemobartonella, Haemophilus, Hafnia, Hahella, Halanaerobacter, Halanaerobium, Haliangium, Haliscomenobacter, Hallella, Haloanaerobacter, Haloanaerobium, Halobacillus, Halobacteroides, Halocella, Halochromatium, Haloincola, Halomicrobium, Halomonas, Halonatronum, Halorhodospira, Halospirulina, Halothermothrix, Halothiobacillus, Halovibrio, Helcococcus, Heliobacillus, Helicobacter, Heliobacterium, Heliophilum, Heliorestis, Heliothrix, Herbaspirillum, Herbidospora, Herpetosiphon, Hippea, Hirschia, Histophilus, Holdemania, Hollandina, Holophaga, Holospora, Hongia, Hydrogenobacter, Hydrogenobaculum, Hydrogenophaga, Hydrogenophilus, Hydrogenothermus, Hydrogenovibrio, Hymenobacter, Hyphomicrobium, Hyphomonas, Ideonella, Idiomarina, Ignavigranum, Ilyobacter, Inquilinus, Intrasporangium, Iodobacter, Isobaculum, Isochromatium, Isosphaera, Janibacter, Jannaschia, Janthinobacterium, Jeotgalibacillus, Jeotgalicoccus, Johnsonella, Jonesia, Kerstersia, Ketogulonicigenium, Ketogulonigenium, Kibdelosporangium, Kineococcus, Kineosphaera, Kineosporia, Kingella, Kitasatoa, Kitasatospora, Kitasatosporia, Klebsiella, Kluyvera, Knoellia, Kocuria, Koserella, Kozakia, Kribbella, Kurthia, Kutzneria, Kytococcus, Labrys, Lachnobacterium, Lachnospira, Lactobacillus, Lactococcus, Lactosphaera, Lamprobacter, Lamprocystis, Lampropedia, Laribacter, Lautropia, Lawsonia, Lechevalieria, Leclercia, Legionella, Leifsonia, Leisingera, Leminorella, Lentibacillus, Lentzea, Leptonema, Leptospira, Leptospirillum, Leptothrix, Leptotrichia, Leucobacter, Leuconostoc, Leucothrix, Levinea, Lewinella, Limnobacter, Limnothrix, Listeria, Listonella, Lonepinella, Longispora, Lucibacterium, Luteimonas, Luteococcus, Lysobacter, Lyticum, Macrococcus, Macromonas, Magnetospirillum, Malonomonas, Mannheimia, Maricaulis, Marichromatium, Marinibacillus, Marinilabilia, Marinilactibacillus, Marinithermus, Marinitoga, Marinobacter, Marinobacterium, Marinococcus, Marinomonas, Marinospirillum, Marmoricola, Massilia, Megamonas, Megasphaera, Meiothermus, Melissococcus, Melittangium, Meniscus, Mesonia, Mesophilobacter, Mesoplasma, Mesorhizobium, Methylarcula, Methylobacillus, Methylobacter, Methylobacterium, Methylocaldum, Methylocapsa, Methylocella, Methylococcus, Methylocystis, Methylomicrobium, Methylomonas, Methylophaga, Methylophilus, Methylopila, Methylorhabdus, Methylosarcina, Methylosinus, Methylosphaera, Methylovorus, Micavibrio, Microbacterium, Microbispora, Microbulbifer, Micrococcus, Microcyclus, Microcystis, Microellobosporia, Microlunatus, Micromonas, Micromonospora, Micropolyspora, Micropruina, Microscilla, Microsphaera, Microtetraspora, Microvirga, Microvirgula, Mitsuokella, Mobiluncus, Modestobacter, Moellerella, Mogibacterium, Moorella, Moraxella, Morganella, Moritella, Morococcus, Muricauda, Muricoccus, Mycetocola, Mycobacterium, Mycoplana, Mycoplasma, Myroides, Myxococcus, Nannocystis, Natroniella, Natronincola, Natronoincola, Nautilia, Neisseria, Neochlamydia, Neorickettsia, Neptunomonas, Nesterenkonia, Nevskia, Nitrobacter, Nitrococcus, Nitrosococcus, Nitrosolobus, Nitrosomonas, Nitrosospira, Nitrospina, Nitrospira, Nocardia, Nocardioides, Nocardiopsis, Nonomuraea, Nonomuria, Novosphingobium, Obesumbacterium, Oceanicaulis, Oceanimonas, Oceanisphaera, Oceanithermus, Oceanobacillus, Oceanobacter, Oceanomonas, Oceanospirillum, Ochrobactrum, Octadecabacter, Oenococcus, Oerskovia, Okibacterium, Oleiphilus, Oleispira, Oligella, Oligotropha, Olsenella, Opitutus, Orenia, Oribaculum, Orientia, Ornithinicoccus, Ornithinimicrobium, Ornithobacterium, Oscillochloris, Oscillospira, Oxalicibacterium, Oxalobacter, Oxalophagus, Oxobacter, Paenibacillus, Pandoraea, Pannonibacter, Pantoea, Papillibacter, Parachlamydia, Paracoccus, Paracraurococcus, Paralactobacillus, Paraliobacillus, Parascardovia, Parvularcula, Pasteurella, Pasteuria, Paucimonas, Pectinatus, Pectobacterium, Pediococcus, Pedobacter, Pedomicrobium, Pelczaria, Pelistega, Pelobacter, Pelodictyon, Pelospora, Pelotomaculum, Peptococcus, Peptoniphilus, Peptostreptococcus, Persephonella, Persicobacter, Petrotoga, Pfennigia, Phaeospirillum, Phascolarctobacterium, Phenylobacterium, Phocoenobacter, Photobacterium, Photorhabdus, Phyllobacterium, Pigmentiphaga, Pilimelia, Pillotina, Pimelobacter, Pirella, Pirellula, Piscirickettsia, Planctomyces, Planktothricoides, Planktothrix, Planobispora, Planococcus, Planomicrobium, Planomonospora, Planopolyspora, Planotetraspora, Plantibacter, Pleisomonas, Plesiocystis, Plesiomonas, Polaribacter, Polaromonas, Polyangium, Polynucleobacter, Porphyrobacter, Porphyromonas, Pragia, Prauserella, Prevotella, Prochlorococcus, Prochloron, Prochlorothrix, Prolinoborus, Promicromonospora, Propionibacter, Propionibacterium, Propionicimonas, Propioniferax, Propionigenium, Propionimicrobium, Propionispira, Propionispora, Propionivibrio, Prosthecobacter, Prosthecochloris, Prosthecomicrobium, Proteus, Protomonas, Providencia, Pseudaminobacter, Pseudoalteromonas, Pseudoamycolata, Pseudobutyrivibrio, Pseudocaedibacter, Pseudomonas, Pseudonocardia, Pseudoramibacter, Pseudorhodobacter, Pseudospirillum, Pseudoxanthomonas, Psychrobacter, Psychroflexus, Psychromonas, Psychroserpens, Quadricoccus, Quinella, Rahnella, Ralstonia, Ramlibacter, Raoultella, Rarobacter, Rathayibacter, Reichenbachia, Renibacterium, Rhabdochromatium, Rheinheimera, Rhizobacter, Rhizobium, Rhizomonas, Rhodanobacter, Rhodobaca, Rhodobacter, Rhodobium, Rhodoblastus, Rhodocista, Rhodococcus, Rhodocyclus, Rhodoferax, Rhodoglobus, Rhodomicrobium, Rhodopila, Rhodoplanes, Rhodopseudomonas, Rhodospira, Rhodospirillum, Rhodothalassium, Rhodothermus, Rhodovibrio, Rhodovulum, Rickettsia, Rickettsiella, Riemerella, Rikenella, Rochalimaea, Roseateles, Roseburia, Roseibium, Roseiflexus, Roseinatronobacter, Roseivivax, Roseobacter, Roseococcus, Roseomonas, Roseospira, Roseospirillum, Roseovarius, Rothia, Rubrimonas, Rubritepida, Rubrivivax, Rubrobacter, Ruegeria, Rugamonas, Ruminobacter, Ruminococcus, Runella, Saccharobacter, Saccharococcus, Saccharomonospora, Saccharopolyspora, Saccharospirillum, Saccharothrix, Sagittula, Salana, Salegentibacter, Salibacillus, Salinibacter, Salinibacterium, Salinicoccus, Salinisphaera, Salinivibrio, Salmonella, Samsonia, Sandaracinobacter, Sanguibacter, Saprospira, Sarcina, Sarcobium, Scardovia, Schineria, Schlegelella, Schwartzia, Sebaldella, Sedimentibacter, Selenihalanaerobacter, Selenomonas, Seliberia, Serpens, Serpula, Serpulina, Serratia, Shewanella, Shigella, Shuttleworthia, Silicibacter, Simkania, Simonsiella, Sinorhizobium, Skermanella, Skermania, Slackia, Smithella, Sneathia, Sodalis, Soehngenia, Solirubrobacter, Solobacterium, Sphaerobacter, Sphaerotilus, Sphingobacterium, Sphingobium, Sphingomonas, Sphingopyxis, Spirilliplanes, Spirillospora, Spirillum, Spirochaeta, Spiroplasma, Spirosoma, Sporanaerobacter, Sporichthya, Sporobacter, Sporobacterium, Sporocytophaga, Sporohalobacter, Sporolactobacillus, Sporomusa, Sporosarcina, Sporotomaculum, Staleya, Staphylococcus, Stappia, Starkeya, Stella, Stenotrophomonas, Sterolibacterium, Stibiobacter, Stigmatella, Stomatococcus, Streptacidiphilus, Streptimonospora, Streptoalloteichus, Streptobacillus, Streptococcus, Streptomonospora, Streptomyces: S. abikoensis, S. erumpens, S. erythraeus, S. michiganensis, S. microflavus, S. zaomyceticus, Streptosporangium, Streptoverticillium, Subtercola, Succiniclasticum, Succinimonas, Succinispira, Succinivibrio, Sulfitobacter, Sulfobacillus, Sulfurihydrogenibium, Sulfurimonas, Sulfurospirillum, Sutterella, Suttonella, Symbiobacterium, Symbiotes, Synergistes, Syntrophobacter, Syntrophobotulus, Syntrophococcus, Syntrophomonas, Syntrophospora, Syntrophothermus, Syntrophus, Tannerella, Tatlockia, Tatumella, Taylorella, Tectibacter, Teichococcus, Telluria, Tenacibaculum, Tepidibacter, Tepidimonas, Tepidiphilus, Terasakiella, Teredinibacter, Terrabacter, Terracoccus, Tessaracoccus, Tetragenococcus, Tetrasphaera, Thalassomonas, Thalassospira, Thauera, Thermacetogenium, Thermaerobacter, Thermanaeromonas, Thermanaerovibrio, Thermicanus, Thermithiobacillus, Thermoactinomyces, Thermoanaerobacter, Thermoanaerobacterium, Thermoanaerobium, Thermobacillus, Thermobacteroides, Thermobifida, Thermobispora, Thermobrachium, Thermochromatium, Thermocrinis, Thermocrispum, Thermodesulfobacterium, Thermodesulforhabdus, Thermodesulfovibrio, Thermohalobacter, Thermohydrogenium, Thermoleophilum, Thermomicrobium, Thermomonas, Thermomonospora, Thermonema, Thermosipho, Thermosyntropha, Thermoterrabacterium, Thermothrix, Thermotoga, Thermovenabulum, Thermovibrio, Thermus, Thialkalicoccus, Thialkalimicrobium, Thialkalivibrio, Thioalkalicoccus, Thioalkalimicrobium, Thioalkalispira, Thioalkalivibrio, Thiobaca, Thiobacillus, Thiobacterium, Thiocapsa, Thiococcus, Thiocystis, Thiodictyon, Thioflavicoccus, Thiohalocapsa, Thiolamprovum, Thiomargarita, Thiomicrospira, Thiomonas, Thiopedia, Thioploca, Thiorhodococcus, Thiorhodospira, Thiorhodovibrio, Thiosphaera, Thiospira, Thiospirillum, Thiothrix, Thiovulum, Tindallia, Tissierella, Tistrella, Tolumonas, Toxothrix, Trabulsiella, Treponema, Trichlorobacter, Trichococcus, Tropheryma, Tsukamurella, Turicella, Turicibacter, Tychonema, Ureaplasma, Ureibacillus, Vagococcus, Vampirovibrio, Varibaculum, Variovorax, Veillonella, Verrucomicrobium, Verrucosispora, Vibrio, Victivallis, Virgibacillus, Virgisporangium, Virgosporangium, Vitellibacter, Vitreoscilla, Vogesella, Volcaniella, Vulcanithermus, Waddlia, Weeksella, Weissella, Wigglesworthia, Williamsia, Wolbachia, Wolinella, Xanthobacter, Xanthomonas, Xenophilus, Xenorhabdus, Xylanimonas, Xylella, Xylophilus, Yersinia, Yokenella, Zavarzinia, Zobellia, Zoogloea, Zooshikella, Zymobacter, Zymomonas, and Zymophilus.
Methods according to aspects of the present disclosure can be used to detect various types of virus including, but not limited to dsDNA viruses (e.g. Adenoviruses, Herpesviruses, Poxviruses); ssDNA viruses (+ strand or “sense”) DNA (e.g. Parvoviruses); dsRNA viruses (e.g. Reoviruses); (+)ssRNA viruses (+ strand or sense) RNA (e.g. Coronaviruses, Picornaviruses, Togaviruses); (−)ssRNA viruses (− strand or antisense) RNA (e.g. Orthomyxoviruses, Rhabdoviruses); ssRNA-RT viruses (+ strand or sense) RNA with DNA intermediate in life-cycle (e.g. Retroviruses); and dsDNA-RT viruses DNA with RNA intermediate in life-cycle (e.g. Hepadnaviruses).
Methods according to aspects of the present disclosure can be used to detect various types of virus, including pathogens and viruses which are not ordinarily pathogenic (e.g. viral vectors for nucleic acid delivery) including, but not limited to viruses of any of the following families: Anelloviridae, Arenaviridae, Arterivirus, Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Closteroviridae, Comoviridae, Coronaviridae, Cystoviridae, Filoviridae, Flaviviridae, Flexiviridae, Hepadnaviridae, Hepevirus, Herpesviridae, Leviviridae, Luteoviridae, Mesoniviridae, Mononegavirales, Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Picobirnaviridae, Picornaviridae, Polyomaviridae, Potyviridae, Poxviridae, Pneumoviridae, Reoviridae, Retroviridae, Rhabdoviridae, Roniviridae, Sequiviridae, Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, and Tymoviridae.
Methods according to aspects of the present disclosure can be used to detect various types of viral infection caused by one or more of: adeno-associated virus, adenovirus, Aichi virus, Alfuy virus, Australian bat lyssavirus, Banna virus, Banzi virus, Barmah forest virus, BK polyomavirus, bovine diarrhea virus, Bunyamwera virus, Bunyavirus La Crosse, Bunyavirus snowshoe hare, Cercopithecine herpesvirus, Chandipura virus, Chikungunya virus, Cosavirus A, Cowpox virus, Coxsackievirus, Crimean-Congo hemorrhagic fever virus, cytomegalovirus, Dengue virus (DNV), Dhori virus, Dugbe virus, Duvenhage virus, Eastern equine encephalitis virus, Ebolavirus, Echovirus, Encephalomyocarditis virus (EMCV), Enterovirus, Epstein-Barr virus, European bat lyssavirus, GB virus C/Hepatitis G virus, Hantaan virus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis delta virus, Human herpesvirus 1, Human herpesvirus 2, Human herpesvirus 6, Human herpesvirus 7, Human herpesvirus 8, human cytomegalovirus (hCMV), Human immunodeficiency virus, Horsepox virus, Ilheus virus, influenza virus, including avian influenza virus, human influenza virus, and swine influenza virus, Influenza A virus, Influenza B virus, Influenza C virus, human papillomavirus 1, human papillomavirus 2, human papillomavirus 16, human papillomavirus 18, Human parainfluenza, Human parvovirus B19, Human spumaretrovirus, Human T-lymphotropic virus, Human torovirus, Isfahan virus, Japanese encephalitis virus, JC polyomavirus, Junin virus, KI Polyomavirus, Kokobera virus, Kunjin virus, Kyasanur forest disease virus, Lagos bat virus, Lake Victoria Marburgvirus, Langat virus, Lassa virus, Lordsdale virus, louping-ill virus, Lymphocytic choriomeningitis virus, Machupo virus, Marburg virus, Mayaro virus, measles virus, Mengo encephalomyocarditis virus, Merkel cell polyomavirus, MERS-coronavirus (MERS), metapneumovirus, Molluscum contagiosum virus, Mokola virus, Monkeypox virus, Mosaic Viruses, Mumps virus, Murray Valley encephalitis virus, New York virus, Nipah virus, norovirus, O'nyong-nyong virus, Orf virus, Oropouche virus, parainfluenza virus, Pichinde virus, poliovirus, Powassan virus, Punta toro phlebovirus, Puumala virus, Rabies virus, respiratory syncytial virus (RSV), rhinovirus, Rift valley fever virus, Rocio virus, Rosavirus A, Ross river virus, Rotavirus A, Rotavirus B, RotavirusC, Rubella virus, Sagiyama virus, Salivirus A, Sandfly fever sicilian virus, Sapporo virus, SARS-coronavirus (SARS), SARS-CoV-2 coronavirus, Semliki forest virus, Seoul virus, Simian foamy virus, Simian virus 5, Sindbis virus, Southampton virus, St. Louis encephalitis virus, Tacaribe virus, Tick-borne powassan virus, tick-borne encephalitis virus, Torque teno virus, Toscana virus, Uukuniemi virus, Vaccinia virus, Varicella-zoster virus, Variola virus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, Western equine encephalitis virus, WU polyomavirus, West Nile virus, Yaba monkey tumor virus, Yaba-like disease virus, yellow fever virus, and Zika virus.
Methods according to aspects of the present disclosure can be used to detect various types of fungal or yeast infection caused by an organism such as, without limitation: Aspergillus, Blastomyces, Candida, Candida auris, Coccidioides, Cryptococcus neoformans, Cryptococcus gatti, Histoplasma, mucormycetes, Pneumocystis jirovecii, Sporothrix, Epidermophyton floccosum, fungi of the genus Trichophyton including Trichophyton rubrum and Trichophyton mentagrophytes, Trichophyton mengninii, Trichophyton schoenleinii, Trichophyton tonsurans, Micosporum canis, Microsporum audouinii, Microsporum gypseum, and Pityrosporum orbicular
Fungal diseases include aspergillosis, blastomycosis, candiasis, coccidiomycosis, cryptococcosis, histoplasmosis, mucorycosis, mycetoma, pneumocystis pneumonia, dermatophytosis, sporotrichosis, paracoccidioidmycosis, pseudallescheriasis, and talaromycosis.
Methods according to aspects of the present disclosure can be used to detect various types of infection caused by a parasitic organism such as, without limitation: protozoans including Sarcodina, Mastigophora, Ciliophora, and Sporozoa; helminths including platyhelminths, acanthocephalins, and nematodes; and ectoparasites, including ticks, fleas, lice, and mites.
Methods according to aspects of the present disclosure can be used to detect various types of infection caused by a parasitic organism such as, without limitation: Cryptosporidium parvum, Cryptosporidium hominis, Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi.
Parasitic diseases include, without limitation, malaria, giardiasis, Babesiosis, cyclosporiasis, cryptosporidiosis, amoebiasis lymphatic filariasis, Leishmaniasis, onchocerciasis, schistosomiasis, Toxoplasmosis, trichomoniasis, trypanosomiasis, and Guinea worm disease, and organisms associated with these or other parasitic diseases can be assayed according to aspects of the present disclosure
A reporter nucleic acid is provided according to aspects of the present disclosure which is capable of being cleaved by “trans” cleavage, also called “collateral” cleavage and “off-target” cleavage of an activated ribonucleoprotein (RNP) complex of a Cas12 or Cas 13 protein and a guide RNA (crRNA) specifically bound to a target nucleic acid, wherein specific binding of the crRNA and the target nucleic acid includes specific binding of complementary nucleic acid sequences.
The term “complementary” as used herein refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen-bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a “percent complementarity” or “percent homology” to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3′-TCGA-5′ is 100% complementary to the nucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′ is 100% complementary to a region of the nucleotide sequence 5′-TAGCTG-3′.
A crRNA oligonucleotide that is specific for a target nucleic acid will specifically hybridize to the target nucleic acid under suitable conditions. As used herein, the terms “hybridize,” “hybridization,” “hybridizing,” or grammatical equivalents thereof refer to the process by which an oligonucleotide single strand anneals with a complementary strand through base pairing under defined hybridization conditions. The terms “specific hybridization,” “specifically hybridize,” “specifically hybridized,” and the like, indicate that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after subsequent washing steps. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may occur, for example, at 65° C. in the presence of about 6×SSC. Stringency of hybridization may be expressed, in part, with reference to the temperature under which the wash steps are carried out. Such temperatures are typically selected to be about 5° C. to 20° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Equations for calculating Tm, for example, nearest-neighbor parameters, and conditions for nucleic acid hybridization are known in the art.
Stringency of hybridization and washing conditions depends on several factors, including the Tm of the probe and target and ionic strength of the hybridization and wash conditions, as is well-known to the skilled artisan. Hybridization and conditions to achieve a desired hybridization stringency are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001; and Ausubel, F. et al., (Eds.), Short Protocols in Molecular Biology, Wiley, 2002. High stringency hybridization conditions are those which only allow hybridization of substantially complementary nucleic acids. Typically, nucleic acids having about 85-100% complementarity are considered highly complementary and hybridize under high stringency conditions. Intermediate stringency conditions are exemplified by conditions under which nucleic acids having intermediate complementarity, about 50-84% complementarity, as well as those having a high degree of complementarity, hybridize. In contrast, low stringency hybridization conditions are those in which nucleic acids having a low degree of complementarity hybridize.
A reporter nucleic acid sequence can be DNA or RNA depending on the Cas protein used. The reporter nucleic acid sequence can be any size or conformation detectable by the nanopore counting device of the present disclosure and capable of being cleaved by the “trans” cleavage activity of the Cas protein used. Typically, the reporter nucleic acid sequence has a size in the range of about 500 nucleotides to about 100,000 nucleotides, such as in the range of about 750 nucleotides to about 50,000 nucleotides, such as in the range of about 900 nucleotides to about 25,000 nucleotides, such as in the range of about 1,000 nucleotides to about 10,000 nucleotides.
A reporter nucleic acid sequence provided according to aspects of the present disclosure is a single-stranded DNA molecule or a single-stranded RNA molecule. The reporter nucleic acid sequence may be linear or circular.
A reporter nucleic acid provided according to aspects of the present disclosure does not include an exogenous label such as a fluorescent label, a chemiluminescent label, a bioluminescent label, a magnetic particle, a radioisotope, or a chromophore.
A target nucleic acid sequence is a nucleic acid sequence of interest and the object of analysis in an assay method according to the present disclosure. The target nucleic acid can be RNA or DNA, depending on the Cas protein used. The target nucleic acid can be genomic DNA of an organism present in, or suspected of being present in, a sample. The target nucleic acid can be RNA transcribed from DNA of an organism present in, or suspected of being present in, a sample. The target nucleic acid can be viral DNA or RNA of a virus present in, or suspected of being present in, a sample. The target nucleic acid can be bacterial DNA or RNA of bacteria present in, or suspected of being present in, a sample. The target nucleic acid can be fungal DNA or RNA of a fungus present in, or suspected of being present in, a sample. The target nucleic acid can be parasite DNA or RNA of a parasite present in, or suspected of being present in, a sample. A target nucleic acid sequence may be selected which is specific for a particular organism to be detected, such as, a species or strain of virus or a species or strain of bacteria. A target nucleic acid sequence may be selected which is common to a number of organisms of a similar type, such as, a genus of virus or a genus of bacteria.
According to aspects, a protospacer adjacent motif (PAM) or PAM-like motif is adjacent the target nucleic acid sequence which directs binding of the non-activated RNP complex to the target nucleic acid to form an activated RNP complex.
The PAM may be a 5′ PAM located upstream of the 5′ end of the target nucleic acid sequence (also known as a protospacer), or a 3′ PAM located downstream of the 5′ end of the target nucleic acid. PAMs are typically 2-5 base pair sequences adjacent the target nucleic acid sequence. For Cas12a, a prototypical PAM is TTTV, where V is A, C, or G, although different Cas 12 enzymes prefer or require particular PAMs. Particular PAMs for particular Cas12 enzymes are known in the art or can be determined using known methodology, see for example, T. Jacobsen et al., Nucleic Acids Research, 48(10): 5624-5638, 2020. Cas13 enzymes typically do not require a PAM.
A CRISPR-Cas system guide RNA (crRNA) is provided according to aspects of the present disclosure that specifically hybridizes to the target nucleic acid sequence. The crRNA contains a target-specific nucleotide sequence (also called a “spacer”) complementary, or substantially complementary, to the target nucleic acid or a region of the target nucleic acid. The target-specific nucleotide sequence of the crRNA contains about 15 to 50 nucleotides, preferably 20 to 25 nucleotides. The target-specific nucleotide sequence of the crRNA contains about 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, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides, or preferably 20, 21, 22, 23, 24, or 25 nucleotides.
According to aspects of the present disclosure, the target-specific nucleotide sequence of the crRNA is substantially complementary to the target sequence, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more complementarity to the target nucleic acid sequence. According to aspects of the present disclosure, there are one or more differences between the target-specific nucleotide sequence of the crRNA and the target nucleic acid sequence, such as a one base pair mismatch between the target-specific nucleotide sequence of the crRNA and the target nucleic acid sequence, such as a two base pair mismatch between the target-specific nucleotide sequence of the crRNA and the target nucleic acid sequence, such as a three base pair mismatch between the target-specific nucleotide sequence of the crRNA and the target nucleic acid sequence, such as a four base pair mismatch between the target-specific nucleotide sequence of the crRNA and the target sequence, or such as a five base pair mismatch between the target-specific nucleotide sequence of the crRNA and the target nucleic acid sequence.
The ability of a crRNA to form a complex with a Cas12 or Cas13 enzyme and form an activated complex with a target nucleic acid sequence may be assessed by any suitable assay. For example, the crRNA, Cas enzyme, and target nucleic acid may be included in a reaction vessel under reaction conditions, followed by an assessment of preferential targeting, such cleavage of the target nucleic acid sequence, assessed by any suitable assay, such as, but not limited to gel electrophoresis. Other assessment methods are possible, and will be recognized by those skilled in the art.
The crRNA further contains a direct repeat (DR) sequence located 5′ (i.e. upstream) or 3′ (i.e. downstream) of the target-specific nucleotide sequence which interacts with the Cas12 or Cas13 protein. Typically a DR sequence is about 18 to 40 nucleotides in length which may form a hairpin stem-loop structure.
A crRNA may be produced by expression using recombinant molecular biology techniques. For example, a crRNA may be designed as a DNA molecule which can be translated to RNA using an in vitro transcription/expression system. A crRNA may also be obtained commercially.
The terms “expression,” “expressing,”. “expresses” and grammatical equivalents refer to transcription of a gene to produce a corresponding mRNA and/or translation of the mRNA to produce the corresponding protein.
A nucleic acid encoding one or more nucleic acid sequences, such as a crRNA, can be cloned into an expression vector for expression of the encoded nucleic acid sequence.
A nucleic acid encoding one or more peptides or proteins, such as a Cas protein, can be cloned into an expression vector for expression of the encoded peptides and/or protein(s).
The term “expression vector” is used to refer to a double-stranded recombinant nucleotide sequence containing a desired coding sequence and containing one or more regulatory elements necessary or desirable for the expression of the operably-linked coding sequence.
Expression vectors can be prokaryotic vectors, e.g., plasmids, or shuttle vectors, insect vectors, or eukaryotic vectors, and expression can be in vitro or in vivo. Suitable expression systems, and associated expression methods, are well known in the art and described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989; 3rd ed., 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel et al., Current Protocols in Molecular Biology. Such nucleic acids and proteins may also be chemically synthesized by well-known methods or may be obtained from commercial sources. Further, kits including suitable expression systems are commercially available.
An included Cas enzyme has “trans” activity to cleave the reporter nucleic acid at least once, thereby changing the size and/or conformation of the reporter nucleic acid.
According to aspects of the present disclosure, a CRISPR/Cas protein is used in methods according to aspects of the present disclosure, wherein the CRISPR/Cas protein includes a Cas protein capable of forming a non-activated ribonucleoprotein (RNP) complex (non-activated RNP) with the guide RNA (crRNA), and wherein the non-activated RNP is capable of binding to the target nucleic acid, thereby forming an activated RNP complex (activated RNP) having “trans” activity to cleave the reporter nucleic acid at least once, thereby changing the size and/or conformation of the reporter nucleic acid, thereby producing a detectable signal indicative of the presence of the target nucleic acid in the sample.
CRISPR/Cas proteins characterized by capability to indiscriminately cleave non-target nucleic acids once “activated” by formation of an activated RNP complex include Type V and Type VI CRISPR/Cas systems, including Cas12 including subtypes V-A, V-B, and variants thereof, and Cas13 including subtypes VI-A, VI-B, VI-C, VI-D (also known as Cas13a, Cas13b, Cas13c, and Cas13d) and variants thereof. Cas13a is also known as C2c2.
CRISPR/Cas12 proteins target single stranded DNA and, once an activated complex is formed including a Cas12 protein, crRNA, and the target RNA, Cas12 proteins indiscriminately cleave non-target single-stranded DNA.
Non-limiting examples of Cas12 proteins used in methods according to aspects of the present disclosure are those from an organism selected from the group consisting of: Acidaminococcus sp., Bacteroidales, Clostridia, Fibrobacteraceae, Lachnospiraceae, Prevotella sp., Spirochaetia, Succinivibrionaceae,
Non-limiting examples of Cas12 proteins used in methods according to aspects of the present disclosure are those from an organism selected from the group consisting of: Acidaminococcus, Agathobacter, Anaerovibrio, Arcobacter, Bacteroides, Butyrivibrio, Flavobacterium, and Treponema Campylobacter, Corynebacter, Eubacterium, Fiihfactor, Flaviivola, Flavobacterium, Francisella, Helcococcus, Oribacterium, Pseudobutyrivibrio, Proteocatella, Sneathia, Sulfuricurvum, Synergistes, and Treponema.
Cas12 proteins have been isolated and characterized from all of the above and include, but are not limited to: Arcobacter butzleri L348 (AbCas12a), Agathobacter rectalis strain 2789STDY5834884 (ArCas12a), Acidaminococcus sp. BV3L6 (AsCas12a), Anaerovibrio sp. RM50 (As2Cas12a), Bacteroidales bacterium KA00251 (BbCas12a), Bacteroidetes oral taxon 274 (BoCas12a), Butyrivibrio sp. NC3005 (BsCas12a), Candidate division WS6 bacterium (C6Cas12a), Coprococcus eutactus, Treponema endosymbiont of Eucomonympha sp. (EsCas12a), Fibrobacter succinogenes, Flavobacterium branchiophilum FL-15 (FbCas12a), Francisella novicida (FnCas12a), Helcococcus kunzii ATCC 51366 (HkCas12a), Lachnospira pectinoschiza strain 2789STDY5834886 (LpCas12a), Lachnospiraceae bacterium (LbCas12a), Candidatus Methanomethylophilus alvus Mx1201 (MaCas12a), Oribacterium sp. NK2B42 (OsCas12a), Candidatus Peregrinibacteria bacterium GW2011 (PbCas12a), Parcubacteria group bacterium GW2011 (PgbCas12a), Proteocatella sphenisci DSM 23131 (PsCas12a), Pseudobutyrivibrio ruminis CF1b (PrCas12a), Pseudobutyrivibrio xylanivorans strain DSM 10317 (PxCas12a), Candidatus Roizmanbacteria bacterium GW2011 (RbCas12a), Sneathia amnii strain SN3 (SaCas12a), Sulfuricurvum sp. PC08-66 (SsCas12a), Synergistes jonesii strain 78-1 (SjCas12a), Succinivibrio dextrinosolvens H5 (SdCas12a), Thiomicrospira sp. XS5 (TsCas12a), and Uncultured bacterium (gcode 4) ACD 3C00058 (U4Cas12a), any of which may be used in methods according to aspects of the present disclosure.
Corynebacterium diphtheria Cas12, 933 aa:
Proteocatella sphenisci Cas12a, 1154 aa:
CRISPR/Cas13 proteins target single-stranded RNA and, once an activated complex is formed including a Cas13 protein, crRNA, and the target RNA, Cas13 proteins indiscriminately cleave non-target single-stranded RNA.
Non-limiting examples of Cas13 proteins used in methods according to aspects of the present disclosure are those from an organism selected from the group consisting of: Azospirillum, Bacteroides, Campylobacter, Corynebacter, Eubacterium, Filifactor, Flaviivola, Flavobacterium, Gluconacetobacter, Lactobacillus, Legionella, Leptotrichia, Listeria, Mycoplasma, Neisseria, Nitratifractor, Parvibaculum, Roseburia, Sphaerochaeta, Staphylococcus, Streptococcus, Sutterella, and Treponema.
Cas13 proteins have been isolated and characterized from the following: Leptotrichia buccalis, Leptotrichia shahii, Leptotrichia wadei, Ruminococcus flavefaciens, Bergeyella zoohelcum, Prevotella buccae, Eubacteriaceae bacterium, Eubacterium rectale, Listeria seeligeri, Carnobacterium gallinarum, Clostridium aminophilum, Herbinix hemicellulosilytics, Lachnospiraceae bacterium, Leptotrichia buccalis, Listeria weihenstephanensis, Listeriaceae bacterium, Paludibacter propionicigenes, Rhodobacter capsulatus, see for example Abudayyeh, Omar O., et al., Nature, 550(7675), p. 280, 2017, any of which may be used in methods according to aspects of the present disclosure.
Leptotrichia wadei (strain F0279, LwaCas13a)
Herbinix hemicellulosilytics HheC2c2, 1285 aa:
Ruminoccocus Flavefaciens Cas13d protein
Cas12 and Cas13 proteins, and their variants, may be produced by recombinant expression using well-known methodologies of molecular biology, or obtained commercially.
Methods and compositions of the present invention are not limited to particular amino acid sequences identified herein and variants of a reference peptide or protein are encompassed.
Variants of a peptide or protein described herein are characterized by conserved functional properties compared to the corresponding peptide or protein.
As disclosed herein, many Cas12 and Cas13 proteins and variants thereof are known and can be used in assays according to aspects of the present disclosure. Variant Cas12 and Cas13 proteins have at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, 96%, 97%, 98%, 99%, or greater identity to a particular reference Cas12 or Cas13 protein and retains the desired functional abilities of a reference Cas12 or Cas13 protein, including capability to form a complex with a crRNA and corresponding target nucleic acid, and to cleave “off-target” nucleic acids including a reporter nucleic acid.
Non-limiting, example amino acid sequences of Cas12 and Cas13 proteins are included herein. Variant of these or other Cas12 and Cas13 proteins have at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, 96%, 97%, 98%, 99%, or greater identity to a particular reference Cas12 or Cas13 protein and retains the desired functional abilities of a reference Cas12 or Cas13 protein, including capability to form a complex with a crRNA and corresponding target nucleic acid, and to cleave “off-target” nucleic acids including a reporter nucleic acid.
Percent identity is determined by comparison of amino acid or nucleic acid sequences, including a reference amino acid or nucleic acid sequence and a putative homologue amino acid or nucleic acid sequence. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions ×100%). The two sequences compared are generally the same length or nearly the same length. A variant may be a naturally-occurring variant of a reference protein such as an ortholog expressed by another organism or species of organism.
The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. Algorithms used for determination of percent identity illustratively include the algorithms of S. Karlin and S. Altshul, PNAS, 90:5873-5877, 1993; T. Smith and M. Waterman, Adv. Appl. Math. 2:482-489, 1981, S. Needleman and C. Wunsch, J. Mol. Biol., 48:443-453, 1970, W. Pearson and D. Lipman, PNAS, 85:2444-2448, 1988 and others incorporated into computerized implementations such as, but not limited to, GAP, BESTFIT, FASTA, TFASTA; and BLAST, for example incorporated in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.) and publicly available from the National Center for Biotechnology Information.
A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, PNAS 87:2264-2268, modified as in Karlin and Altschul, 1993, PNAS. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches are performed with the NBLAST nucleotide program parameters set, e.g., for score=100, word length=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present invention. BLAST protein searches are performed with the XBLAST program parameters set, e.g., to score 50, word length=3 to obtain amino acid sequences homologous to a protein molecule of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST are utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI BLAST is used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) are used. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 is used.
The percent identity between two sequences is determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
One of skill in the art will recognize that one or more amino acid mutations can be introduced without altering the functional properties of a given nucleic acid or protein, respectively. Mutations can be introduced using standard molecular biology techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis, to produce variants. For example, one or more amino acid substitutions, additions, or deletions can be made without altering the functional properties of a reference protein.
Conservative amino acid substitutions can be made or may be present in reference proteins to produce or identify variants.
Conservative amino acid substitutions are art recognized substitutions of one amino acid for another amino acid having similar characteristics. For example, each amino acid may be described as having one or more of the following characteristics: electropositive, electronegative, aliphatic, aromatic, polar/nonpolar, hydrophobic and hydrophilic. A conservative substitution is a substitution of one amino acid having a specified structural or functional characteristic for another amino acid having the same characteristic. Acidic amino acids include aspartate, glutamate; basic amino acids include histidine, lysine, arginine; aliphatic amino acids include isoleucine, leucine and valine; aromatic amino acids include phenylalanine, tyrosine and tryptophan; polar amino acids include aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine and tyrosine; and hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine and tryptophan; and conservative substitutions include substitution among amino acids within each group. Amino acids may also be described in terms of relative size; alanine, cysteine, aspartate, glycine, asparagine, proline, threonine, serine, valine are all typically considered to be small.
A variant can include synthetic amino acid analogs, amino acid derivatives and/or non-standard amino acids, illustratively including, without limitation, alpha-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy-phenylalanine, djenkolic acid, homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, and ornithine.
A non-activated RNP is formed by contacting a crRNA and Cas 12 or Cas 13 protein. The non-activated RNP is then contacted with the target nucleic acid sequence under conditions allowing specific binding of the non-activated RNP to the target nucleic acid sequence, forming an activated RNP. The activated RNP indiscriminately cleaves the reporter nucleic acid.
According to aspects of the present disclosure, an assay for a target nucleic acid analyte can be quantitative or qualitative. A quantitative assay provides a count of the number of target nucleic acid molecules in a sample, whereas a qualitative assay provides a positive/negative result indicating presence/absence of a target nucleic acid at a specified % confidence level within a specified time.
As described herein, the time needed to determine the presence/absence of a target nucleic acid with a particular confidence level, such as 95% confidence level, or lower, or higher confidence level can be calculated. For any fixed nanopore reading time Tm, one can solve for the maximal λp and therefore the minimal required reaction time Tr.
Alternatively, the assay time can be increased or decreased and concentrations of activated RNP and target nucleic acid adjusted accordingly.
According to aspects of the present disclosure, the non-activated RNP concentration is in a range of about 1 nM-10 uM, such as 10-100 nM. The components of the non-activated RNP are present in a ratio of at least 1:1 such that each component is also present in a concentration of about 1 nM-10 uM, such as 10-100 nM.
Nucleic acid reporter concentration is in the range of about 10 picomolar (pM) to about 1 micromolar (μM), such as about 50 pM to about 250 nanomolar (nM).
According to aspects of the present disclosure, an assay for detection of a nucleic acid analyte includes: 1) formation of a non-activated RNP complex; 2) formation of an activated RNP complex; and 3) indiscriminate cleavage of a nucleic acid reporter in the presence of the activated RNP complex.
Formation of the non-activated RNP complex is achieved by contacting a Cas12 or Cas13 protein with a crRNA under appropriate conditions. Appropriate conditions for formation of a non-activated RNP complex are typically “physiological” conditions, such as in an aqueous medium containing physiological salt concentrations, pH, and at a temperature in the range of about 4° C. to about 37° C., preferably “room temperature” (about 25° C.) to about 37° C., depending on the desired time for formation of the non-activated RNP complex. Phosphate-buffered saline (1×PBS) is a non-limiting example of a suitable aqueous medium containing physiological salt concentrations and physiological pH. At room temperature, non-activated RNP complex is formed in about 5 minutes to about 60 minutes, such as about 15 minutes to about 30 minutes.
Formation of activated RNP complex is achieved by mixing the non-activated RNP with target nucleic acid, double-stranded target DNA for Cas12 and single-stranded target RNA for Cas13 under appropriate conditions.
Appropriate conditions for formation of a non-activated RNP complex are typically “physiological” conditions, such as in an aqueous medium containing physiological salt concentrations, pH, and at a temperature in the range of about 4° C. to about 37° C., preferably “room temperature” (about 25° C.) to about 37° C., depending on the desired time for formation of the non-activated RNP complex. Phosphate-buffered saline (1×PBS) is a non-limiting example of a suitable aqueous medium containing physiological salt concentrations and physiological pH. At 37° C., non-activated RNP complex is formed in about 5 minutes to about 60 minutes, such as about 10 minutes to about 20 minutes.
The nucleic acid reporter is then incubated with the activated RNP complex under appropriate conditions to produce indiscriminate cleavage of nucleic acids by the “off-target” activity of the activated RNP complex, which includes cleavage of the nucleic acid reporter. Appropriate conditions for cleavage include an aqueous Cas enzyme activity-compatible medium containing a divalent cation, preferably Mg++, in an amount sufficient for Cas activity, such as about 1-10 mM MgCl2, such as about 2.5-75 mM MgCl2, such as about 5 mM MgCl2; a salt, such as NaCl or KCl, in a concentration in the range of at least 1 mm and less than 200 mM, a buffer compatible with cleavage activity such as, but not limited to HEPES, substantially protein free, a pH in the range appropriate for the particular buffer, generally in the range of about pH 5 to about pH 9, such as about pH 6 to about pH 8, and at a temperature in the range of about 4° C. to about 37° C., preferably “room temperature” to about 37° C., depending on the desired time for cleavage of the reporter if the target nucleic acid is present. A non-limiting example of a aqueous Cas enzyme activity-compatible medium includes about 40-200 mM NaCl, such as 50-150 mM NaCl, such as 75-100 mM NaCl; about 1-10 mM MgCl2, such as about 2.5-75 mM MgCl2, such as about 5 mM MgCl2; about 10-30 mM HEPES buffer, such as about 20 mM HEPES buffer, about 0.05-5 mM EDTA, such as about 0.1-0.2 mM EDTA, at a pH of about 6.5 at 25° C. At 37° C., the cleavage reaction time can be in the range of about 1 minute to about 60 minutes, such as about 5 minutes to about 30 minutes.
The reaction is then terminated, for example by adding sufficient salt to terminate the reaction and such that the final salt concentration is about 0.5 M to 50 M, for efficient nanopore counting of the remaining nucleic acid reporter. Thus, the reaction is terminated by adding a high concentration of salt, such as KCl or NaCl, such that the final salt concentration is in the range 0.5 M to 50 M, thereby stopping the reaction and providing an ionic environment for the nanopore detection.
Results show that the reporter cleavage rate constant is proportional to the target nucleic acid concentration.
Cleavage of the reporter nucleic acid reduces passage of the reporter nucleic acid through the nanopore such that a reduction of resistive pulses is produced which provides a signal representative of presence of the target nucleic acid in the sample.
Embodiments of inventive compositions and methods are illustrated in the following examples. These examples are provided for illustrative purposes and are not considered limitations on the scope of inventive compositions and methods.
Sequence-Specific Recognition of Virus DNA with Solid-State CRISPR-Cas12a-Assisted Nanopores (SCAN)
Materials and Methods
A.s.Cas12a Ultra (#10001272) and IDTE pH7.5 buffer (#11-01-02-02) were purchased from Integrated DNA Technologies (IDT). dsDNA target nucleic acid and crRNA were also synthesized and purchased from IDT. M13mp18 ssDNA (#N4040S) and NEBuffer 3.1 (#B7203S) were purchased from NEW ENGLAND Biolabs Inc. (NEB). DNA elution buffer was purchased from Zymo Research (#D4036-5). Nuclease-free molecular Biology grade water was purchased from Hyclone (SH30538). DPBS was purchased from Thermo Fisher (#14190250). DNA gel loading dye (6×) was purchased from Thermo Fisher (#R0611). 10× IDT reaction buffer (200 mM HEPES, 1 M NaCl, 50 mM MgCl2 and 1 mM EDTA, PH6.5@25° C.) was made at the lab. MgCl2, NaCl, KCl and Tris-EDTA-buffer solution (10 mM Tris-HCl and 1 mM EDTA) were purchased from Sigma-Aldrich. HEPES was purchased from Gibco #15630-080. Agarose was purchased from Bio-rad (#1613102). Ethidium Bromide (EB) was purchased from Life Technologies (#15585011). DNA Ladder was purchased from Thermo Scientific (#SM0311). Ag/AgCl wires electrodes were fabricated by using 0.2 mm Ag wires (Warner Instruments, Hamden, USA). Micro injectors of 34 gauge were purchased from World Precision Instruments. Piranha solution was made by mixing concentrated sulfuric acid (H2SO4) with hydrogen peroxide (H2O2). Quartz capillaries with inner and outer diameter of 0.5 mm and 1 mm were purchased from Sutter Instrument.
Virus Detection Assay
Aligned sequences of three domains of HIV-1, which are GAG (capsid protein), POL (protease, reverse transcriptase and integrase), and ENV (glycoprotein) were obtained from an HIV sequence database detailed in Rouzioux, C. et al., Retrovirology 2018, 15(1), 30. Shannon entropy value of each nucleotide of the aligned sequences was calculated with the “Entropy-One” function in the HIV sequence database. Two HIV-1 DNA oligonucleotides from the GAG region (HIV-1 Target 1 and Target 2) and two specific crRNAs were synthesized by IDT. Sequences used in this example are as follows:
Target 1: 5′-tatcacctagaacTTTAAATGCATGGGTAAAAGTAGTAgaagagaaggct-3′ (SEQ ID NO:1). The underlined portion is the PAM, upper case is the target nucleic acid sequence, lower case sequence is included to show context in the virus genome.
crRNA1: 5′-UAAUUUCUACUCUUGUAGAUAAUGCAUGGGUAAAAGUAGUA-3′ (SEQ ID NO:2) In this sequence, the first 20 nt, UAAUUUtCUACUCUUGUAGAU (SEQ ID NO:5), constitute the direct repeat, and the remainder, AAUGCAUGGGUAAAAGUAGUA (SEQ ID NO:6), is the target-specific nucleic acid sequence.
Target 2: 5′-ccTTTAACTTCCCTCAGGTCACTCTTTGGcaacgacccctcgtcacaataa-3′ (SEQ ID NO:3). The underlined portion is the PAM, upper case is the target nucleic acid sequence, lower case sequence is included to show context in the virus genome.
crRNA2: 5′-UAAUUUCUACUCUUGUAGAUACUUCCCUCAGGUCACUCUUUGG-3′ (SEQ ID NO:4) In this sequence, the first 20 nt, UAAUUUCUACUCUUGUAGAU (SEQ ID NO:5), constitute the direct repeat, and the remainder, ACUUCCCUCAGGUCACUCUUUGC (SEQ ID NO:7), is the target-specific nucleic acid sequence.
The synthesized HIV-1 DNA oligonucleotides were resuspended in molecular biology grade water, annealed in DNA elution buffer. The crRNAs were resuspended in IDTE pH 7.5 buffer and stored in −80° C. For ribonucleoprotein (RNP) complex formation, Cas12a and crRNA were mixed in 1×PBS to form the non-activated RNP complex at room temperature for 20 min and stored in −80° C. In the cleavage reaction, non-activated RNP complex was mixed with dsDNA target and incubated at 37° C. for 10 min for RNP activation, producing activated RNP complex. Then ssDNA reporters were added and incubated at 37° C. for cleavage. After the reaction, results were examined both in agarose gel and in the nanopore device. For gel imaging, reactions were terminated with DNA loading dye (6×). The 24 μl mixture was loaded to EB-stained 1% (wt/vol) agarose gel for electrophoresis analysis. For nanopore analysis, reactions were terminated by adjusting the salt concentrations to 1M KCl.
Glass Nanopore Fabrication
To remove organic residues from quartz capillaries, as-purchased quartz capillaries were firstly cleaned in Piranha solution for 30 minutes, then rinsed with DI water, and dried in a vacuum oven at 120° C. for 15 min. A two-line recipe, (1) Heat 750, Filament 5, Velocity 50, Delay140, and Pull 50; (2) Heat 710, Filament 4, Velocity 30, Delay 155, and Pull 215, was used to pull the capillaries with a laser pipette puller (P-2000, Sutter Instruments, USA). This recipe typically produces nanopores of diameter around 10 nm.
Nanopore Sensing and Data Analysis
A constant voltage was applied across the glass nanopore by 6363 DAQ card (National Instruments, USA). A transimpedance amplifier (Axopatch 200B, Molecular Device, USA) was used to amplify the resulting current and then digitalized by the 6363 DAQ card at 100 kHz sampling rate. Finally, a customized MATLAB (MathWorks) software was used to analyze the current time trace and extract the single molecule translocation information.
Results
In this example of a SCAN method, circular ssDNAs (M13mp18, 7249 bases) of a known concentration (typically 100 pM) were used as reporters. If target HIV-1 DNAs exist in the analyte solution, the Cas12a/crRNA complex (i.e., non-activated RNP) is activated by binding specifically to the target HIV-1 DNAs, producing an activated RNP complex, see
It is noted that, while the remaining ssDNA reporter can be readily visualized by conventional gel electrophoresis in this example, the nanopore readout is much more sensitive and can be performed in-situ, see
To ensure all events observed in the nanopore sensors correspond to the ssDNA reporter rather than interfering background molecules (e.g., RNPs), a control nanopore experiment was performed using a pure RNP and HIV-1 DNA sample, 30 nM each, without any ssDNA reporter. Not even a single event was observed for a measurement time of 1000 seconds, see
HIV-1 Assay and Buffer Optimization
For sequence-specific recognition of HIV-1 DNA, the crRNAs targeted conserved regions in all HIV-1 subtypes. This example focused on three commonly evaluated domains of HIV-1, which are GAG (capsid protein), POL (protease, reverse transcriptase and integrase), and ENV (glycoprotein), detailed for example in Zhao, J. et al., European Journal of Clinical Microbiology & Infectious Diseases 2019, 38 (5), 829-842; Schlatzer, D. et al., Analytical chemistry 2017, 89 (10), 5325-5332; and Waheed, A. A. et al., AIDS research and human retroviruses 2012, 28 (1), 54-75. Two 50 bp dsDNAs from the GAG region were synthesized as HIV-1 targets. Two specific crRNAs were designed for each of these dsDNAs targets as shown herein.
Three candidate buffers were tested: NEBuffer 3. 1, PBS buffer and IDT buffer, see Table 2 for detailed compositions.
A gel analysis was performed to validate each of these buffers, and results are shown in
It was hypothesized that a buffer with Mg2+ ions that has no BSA is desirable for the SCAN device. The IDT buffer is such a candidate. Functionality of IDT buffer, containing Mg2+ ions and no BSA, in a nucleic acid assay according to aspects of the present disclosure was determined in this example, and results are shown in
Nanopore Event Rate for Circular ssDNA Reporter Quantification
For a typical SCAN experiment, the RNP concentration remains constant during the Cas12a cleavage reaction. To validate if the nanopore event rate can be used as a quantitative readout for the ssDNA reporter concentration at the constant RNP background, nanopore counting experiments with serially diluted ssDNA reporter were performed. In all experiments, the RNP and salt concentration was fixed as 30 nM and 1 M, respectively.
Virus Nucleic Acid-Activated Cas12a Trans-Cleavage Monitored by Nanopore Counting
Having shown the linear relationship between the ssDNA reporter and the nanopore event rate under the constant RNP, an assay for viral target nucleic acid was performed using a SCAN method and apparatus according to aspects of the present disclosure.
Three different HIV-1 target nucleic acid concentrations, 15, 30, and 60 nM, were tested by adding dsDNA sample to the RNP solutions in the IDT buffer. In all the experiments, the initial ssDNA reporter concentration was fixed at 100 pM. The reaction was terminated at various reaction times, 0, 5, 10, 20, and 30 minutes, by adding KCl salt to the final salt concentration of 1M. The remaining ssDNA reporter concentration was measured by the calibration-free nanopore counting method as described below under the heading “Linking ssDNA reporter concentration with translocation rate.”
Linking ssDNA Reporter Concentration with Translocation Rate
In the diffusion-limited region, the capture rate for the conical-shaped glass nanopore is given by:
α=2πμ{circumflex over (d)}ΔV
where μ is the free solution electrophoretic mobility, ΔV is the applied electric potential across the pore, and {circumflex over (d)} is the characteristic length of the nanopore. Also, the baseline current can be estimated as:
I
b=2πΛCion{circumflex over (d)}ΔV
where Λ is the molar conductivity which depends on the mobility and valance of the ions as:
Λ=ΣiNAeziμi
Thus, the ssDNA reporter translocation rate R=αNACDNA can be written as:
where NA is the Avogadro constant and CDNA is the ssDNA reporter concentration. Hence, the remaining ssDNA reporter concentration can be estimated based on their translocation rate through the nanopore:
Statistical Modeling for Qualitative Positive/Negative Test in SCAN
The translocation of molecules through the nanopore is a Poisson process. Thus inferring the event rate from observing n events in Tm will have an uncertainty of (1.96(n)1/2)/Tm. Thus, longer reaction time Tr and measurement time Tm is preferred to make a statistically confident call for a qualitative positive/negative test. However, minimizing the total experimental time (Tr+Tm) would be highly desirable towards a fast sample-to-result turnaround.
In order to estimate the total experimental time for a qualitative positive/negative test, a statistical model was developed. For the negative case (i.e., no reporter degradation), the expected number of events in the nanopore in a measurement time of Tm is given by
λn=αμC0Tm
where μ is the electrophoretic mobility of the ssDNA reporter, α is a constant, and C0 is the initial ssDNA reporter concentration before the reaction. For the positive case after reaction time Tr, the initial reporter concentration C0 would decrease to C0e−kT
λ=αμC0e−kT
in which k is the rate constant that is linearly proportional to the activated RNP concentration. The activated RNP concentration is limited by the smaller values between HIV-1 DNA and RNP concentration in the system and can be written as
k=A×min(CHIV,CRNP)
where A is a constant (0.00148 min−1 nM−1, see Michaelis-Menten kinetics below).
Michaelis-Menten Kinetics
Michaelis-Menten kinetics model describes the relationship between the reaction rate v and the concentration of a substrate C as:
where Vm and Km represent the maximum rate achieved by the system and substrate concentration at which the reaction rate is half of Vm. respectively. For small concentrations, it can be assumed that Km+C=Km:
By introducing the rate constant
C=C
0
e
−kt
Rate constant k is proportional to activated RNP concentration. For this calculation, the concentration of the activated RNP is assumed to be constant. This is a reasonable assumption because the exemplified assay has three steps. (1) RNP formation, Cas12a, and crRNA were mixed in 1×PBS to form the non-activated RNP at room temperature for 20 min. (2) Non-activated RNP complex was mixed with dsDNA target and incubated at 37° C. for 10 min for RNP activation. (3) Then ssDNA reporters were added and incubated at 37° C. for different times (5, 10, 20, and 30 minutes) for cleavage. In step 2, more dsDNA targets were added to the mix to make sure all the RNP complexes have been activated. As a result, the concentration of the activated Cas12a enzyme was constant when the cleavage process starts.
The rate constants at different activated RNP concentrations were obtained by line fitting to the experimental results of 0, 15, 30, and 60 nM, see
It was found that, while the RNP complexes do not produce measurable events, they do affect the electrophoretic mobility of the ssDNA reporters. It was found the reporter electrophoretic mobility reduces exponentially as the RNP concentration is increased, see “RNP affects the reporter electrophoretic mobility” below.
RNP Affects the Reporter Electrophoretic Mobility
While the RNP complexes do not produce measurable events, they do affect the electrophoretic mobility of the ssDNA reporters. To investigate the effect of RNP concentration on the electrophoretic mobility of the reporter, a nanopore experiment on 100 pM ssDNA reporters was performed at four RNP concentrations, 0, 15, 30, and 60 nM. These RNP were not activated, i.e., no target dsDNA. Based on the time traces of the ionic current in
To extract the effective electrophoretic mobility of the ssDNA reporter, a calibration-free nanopore counting method was employed, described herein, to relate the mobility to the molecular event rate and the ionic current baseline. The reduced ssDNA reporter mobility at increased RNP concentration, see
This phenomenon is described by the Ogston-Morris-Rodbard-Chrambach model, in which molecule electrophoretic mobility has an exponential relationship with the obstacles concentration (μ∝e−C
μ=μ0e−βC
where μ0 (1.73×10−8 m2 V−1 s−1) and β (0.025 nM−1) are the constants of the fitted exponential curve to the experimental results, see Ogston-Morris-Rodbard-Chrambach (OMRC) model below.
Ogston-Morris-Rodbard-Chrambach (OMRC) Model
In the framework of the OMRC model, the low-field reduced mobility μ*(CRNP) of an analyte is assumed to be equal to its free available volume f(CRNP):
where μ0 is the free (no obstacle) mobility, and CRNP is the non-activated RNP (obstacle concentration). For a specific system where analytes are considered to be spherical particles, the fractional volume available to the analyte is given by f(CRNP)=e−βC
μ=μ0e−βC
where extracted μ0 and β from the fitting exponential line are 1.73×10−8 m2 V−1 s−1 and 0.025 nM−1, respectively, see
Experimentally observed events for negative and positive case would follow the Poisson distribution with expected values of λn and μp respectively. As illustrated in
Sequence-Specific Example—HIV
In this example, two sets of HIV-1 DNA targets and assays were used, in which each assay was specific to its target, Assay 1 was specific to Target 1 and Assay 2 was specific to Target 2. To test the cross-reactivity of designed assays, gel analysis was performed on the assay products,
Sequence-Specific Example—SARS-Cov-2
In this example, to detect SARS-CoV-2 virus, SARS-CoV-2 virus genome RNA is first converted to DNA and amplified via reverse transcription-recombinase polymerase amplification (RT-RPA) or RT-Loop-mediated isothermal amplification (RT-LAMP). The following PCR primers can be used to amplify SARS-CoV-2 virus nucleic acid of a sample prior to nanopore detection analysis: Forward: ttacaaacattggccgcaaa (SEQ ID NO:8), Reverse: gcgcgacattccgaagaa (SEQ ID NO:9)
Then A.s.Cas12a Ultra (IDT #10001272) Cas12a enzyme with its corresponding crRNA (cccccagcgcttcagcgttc (SEQ ID NO:10) with a PAM tttg) targeting SARS-CoV-2 N gene are contacted to form a non-activated RNP complex by incubation in 1×PBS at room temperature for 20 min. The non-activated RNP complex is then mixed with the amplified samples and incubated at 37° C. for 10 min for RNP activation to form activated RNP complex. A ssDNA reporter is then included in the mixture and incubated at 37° C. for cleavage of the reporter if SARS-CoV-2 N gene target nucleic acid. A solution of high salt concentration, over 1M, is then added to stop the reaction. This mixture is then loaded into the first chamber of a nanopore system as described herein for detection. Correct recognition of SARS-CoV-2 virus will indiscriminately cut the reporter nucleic acids, yielding fewer reads in the nanopore system.
Items
Item 1. A method of detecting an analyte in a solution, comprising: providing a nanopore counting device comprising; a first chamber and a second chamber; a barrier having a nanopore opening defined therein, the barrier separating the first chamber from the second chamber; a control/sensing system operable to apply an electrical potential between a solution in the first chamber and a solution in the second chamber and to sense a voltage and/or current between the chambers; disposing an ion-containing solution in the first and second chambers; disposing a sample in the first chamber, the sample containing or suspected of containing the analyte; applying an electrical potential between the solution in the first chamber and the solution in the second chamber; and sensing a baseline current between the chambers and detecting resistive pulses, wherein detecting resistive pulses provides a signal representative of presence of the analyte in the sample, and wherein no calibration step is performed before disposing the sample.
Item 2. The method of item 1, wherein sensing a baseline current between the chambers and detecting resistive pulses provides a count of a number of molecules of analyte that pass through the nanopore during a period of time.
Item 3. The method of item 2, further comprising determining an estimated concentration of the molecules of analyte in the first chamber, wherein determining comprises calculation using the formula:
where:
Λ is the molar conductivity of the buffer solution in Siemens per meter per mole;
R is the translocation rate in resistive pulses per second;
μ is the free solution electrophoretic mobility of the analyte in meters per volt second;
NA is the Avogadro constant; and
Ib is the baseline current in nA.
Item 4. The method according to any of items 1 to 3, wherein the barrier with the nanopore is a solid state nanopore barrier.
Item 5. The method according any of items 1 to 4, wherein the nanopore has an opening size larger than a size of an analyte molecule, the nanopore opening size being within one order of magnitude of the size of the analyte molecule.
Item 6. The method according to any of items 2 to 5, wherein the period of time is determined by the number of resistive pulse, the number of resistive pulse during the period of time being at least 100.
Item 7. The method according to any of items 1 to 6, wherein the number of resistive pulses is at least 200.
Item 8. The method according to any of items 2 to 7, wherein the number of resistive pulses during the period of time is at least 200.
Item 9. The method according to any of items 1 to 8, wherein the analyte is a target nucleic acid sequence, and further comprising: providing a reporter nucleic acid; providing a CRISPR-Cas system guide RNA (crRNA) that hybridizes to the target nucleic acid sequence; providing a CRISPR enzyme, wherein the CRISPR enzyme is a Cas enzyme capable of forming a non-activated ribonucleoprotein (RNP) complex (non-activated RNP) with the guide RNA, and wherein the non-activated RNP complex is capable of binding to the target nucleic acid sequence, forming an activated RNP complex (activated RNP) having “trans” activity to cleave the reporter nucleic acid; contacting the crRNA and Cas enzyme, thereby forming the non-activated RNP; disposing the non-activated RNP in the first chamber with the sample, wherein the non-activated RNP and the target nucleic acid sequence specifically bind if the target nucleic acid is present in the sample, forming an activated RNP, wherein the activated RNP cleaves the reporter nucleic acid, and wherein cleavage of the reporter nucleic acid reduces passage of the reporter nucleic acid through the nanopore such that a reduction of resistive pulses is produced which provides a signal representative of presence of the target nucleic acid sequence in the sample.
Item 10. The method according to item 9, wherein the target nucleic acid sequence is DNA and the Cas enzyme is a Cas12 enzyme.
Item 11. The method according to item 9, wherein the target nucleic acid sequence is RNA and the Cas enzyme is a Cas13 enzyme.
Item 12. The method according to any of items 9 to 11, wherein the reporter nucleic acid is a linear or circular single-stranded DNA molecule.
Item 13. The method according to any of items 9 to 12, wherein the reporter nucleic acid does not include a label.
Item 14. The method according to any of items 9 to 13, wherein the target nucleic acid sequence is a nucleic acid of a microorganism.
Item 15. The method of any of items 9 to 14, wherein the target nucleic acid sequence is a nucleic acid of a virus, a bacterium, a fungus, or a parasite.
Item 16. The method of any of items 9 to 15, wherein the sample is obtained from a mammal.
Item 17. The method of any of items 9 to 16, wherein the sample is derived from a human.
Item 18. The method of any of items 9 to 17, wherein the sample is derived from a mammal having, or suspected of having, an infection by a virus, a bacterium, a fungus, or a parasite.
Item 19. The method of any of items 9 to 18, wherein the sample is derived from a human having, or suspected of having, an infection by a virus, a bacterium, a fungus, or a parasite.
Item 20. The method of any of items 9 to 15, wherein the sample is an environmental sample, containing, or suspected of containing, a virus, a bacterium, a fungus, or a parasite.
Item 21. The method of any of items 9 to 20, wherein the target nucleic acid sequence is a nucleic acid of a human immunodeficiency virus.
Item 22. The method of any of items 9 to 20, wherein the target nucleic acid sequence is a nucleic acid of a coronavirus.
Item 23. The method of item 22, wherein the coronavirus is a Sars-Cov-2 coronavirus.
Item 24. The method of any of items 9 to 15, wherein the sample is derived from a plant.
Item 25. The method of any of items 9 to 15 or 24, wherein the sample is derived from a plant having, or suspected of having, an infection by a virus, a bacterium, a fungus, or a parasite.
Item 26. A method of detecting a target nucleic acid sequence in a solution, comprising: providing a nanopore counting device comprising; a first chamber and a second chamber; a barrier having a nanopore opening defined therein, the barrier separating the first chamber from the second chamber; a control/sensing system operable to apply an electrical potential between a solution in the first chamber and a solution in the second chamber and to sense a voltage and/or current between the chambers; calibrating the nanopore counting device to determine a rate of translocation of molecules of a calibrant from a calibration solution through the nanopore when a calibrating electrical potential is applied between the chambers; disposing an ion-containing solution in the first and second chambers; providing a reporter nucleic acid; providing a CRISPR-Cas system guide RNA (crRNA) that hybridizes to the target nucleic acid sequence; providing a CRISPR enzyme, wherein the CRISPR enzyme is a Cas enzyme capable of forming a non-activated ribonucleoprotein (RNP) complex (non-activated RNP) with the guide RNA, and wherein the non-activated RNP is capable of binding to the target nucleic acid sequence, forming an activated RNP complex (activated RNP) having “trans” activity to cleave the reporter nucleic acid; contacting the crRNA and Cas enzyme, thereby forming the non-activated RNP; disposing the non-activated RNP in the first chamber with the sample, wherein the non-activated RNP and the target nucleic acid sequence specifically bind if the target nucleic acid is present in the sample, forming an activated RNP, wherein the activated RNP cleaves the reporter nucleic acid; applying an electrical potential between the solution in the first chamber and the solution in the second chamber; sensing current between the first chamber and the second chamber and detecting resistive pulses, wherein cleavage of the reporter nucleic acid reduces passage of the reporter nucleic acid through the nanopore such that a reduction of resistive pulses is produced which provides a signal representative of presence of the target nucleic acid sequence in the sample
Item 27. The method according to item 26, wherein:
the step of detecting resistive pulses further comprises counting resistive pulses to determine a number of reporter nucleic acid molecules that pass through the nanopore during a period of time, thereby determining a rate of translocation of the reporter nucleic acid molecules.
Item 28. The method according to item 26, further comprising determining the estimated concentration of the target nucleic acid sequence in the first chamber based on the reporter nucleic acid translocation rate as compared to the calibrant translocation rate.
Item 29. The method according to any of items 26 to 28, wherein the calibrating step comprises: disposing an ion-containing solution in the first and second chamber and a known concentration of calibrant molecules in the first chamber, the calibrant molecules being the same or similar to the reporter nucleic acid molecules; applying the calibrating electrical potential between the chambers; sensing current between the chambers and counting resistive pulses to determine a number of molecules of the calibrant that pass through the nanopore during a period of time; and determining a rate of translocation for the known concentration of calibrant at the calibrating electrical potential.
Item 30. The method according to any of items 26 to 29, wherein the barrier with the nanopore barrier is a solid state nanopore barrier or a biological nanopore barrier.
Item 31. The method according to any of items 26 to 30, wherein the calibrant molecules are the same as the reporter nucleic acid molecules, the calibrating electrical potential is in the range of 0.5 to 2 times the electrical potential used after the calibrating step, and the ion-containing solution during is the same during the calibrating step and after the calibrating step.
Item 32. The method according to any of items 26 to 31, wherein the target nucleic acid sequence is DNA and the Cas enzyme is a Cas12 enzyme.
Item 33. The method any of items 26 to 31, wherein the target nucleic acid sequence is RNA and the Cas enzyme is a Cas13 enzyme.
Item 34. The method any of items 26 to 33, wherein the reporter nucleic acid is a circular or linear single-stranded DNA molecule.
Item 35. The method any of items 26 to 34, wherein the reporter nucleic acid does not include a label.
Item 36. The method any of items 26 to 35, wherein the target nucleic acid sequence is a nucleic acid of a microorganism.
Item 37. The method of any of items 26 to 36, wherein the target nucleic acid sequence is a nucleic acid of a virus, a bacterium, a fungus, or a parasite.
Item 38. The method of any of items 26 to 37, wherein the sample is obtained from a mammal.
Item 39. The method of any of items 26 to 38, wherein the sample is derived from a human.
Item 40. The method of any of items 26 to 39, wherein the sample is derived from a mammal having, or suspected of having, an infection by a virus, a bacterium, a fungus, or a parasite.
Item 41. The method of any of items 26 to 40, wherein the sample is derived from a human having, or suspected of having, an infection by a virus, a bacterium, a fungus, or a parasite.
Item 42. The method of any of items 26 to 37, wherein the sample is an environmental sample, containing, or suspected of items, a virus, a bacterium, a fungus, or a parasite.
Item 43. The method of any of items 26 to 42, wherein the target nucleic acid sequence is a nucleic acid of a human immunodeficiency virus.
Item 44. The method of any of items 26 to 42, wherein the target nucleic acid sequence is a nucleic acid of a coronavirus.
Item 45. The method of item 44, wherein the coronavirus is a Sars-Cov-2 coronavirus.
Item 46. The method of any of items 26 to 37, wherein the sample is derived from a plant.
Item 47. The method of any of items 26 to 37 or 46, wherein the sample is derived from a plant having, or suspected of having, an infection by a virus, a bacterium, a fungus, or a parasite.
Any patents or publications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication is specifically and individually indicated to be incorporated by reference.
The compositions and methods described herein are presently representative of preferred embodiments, exemplary, and not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art. Such changes and other uses can be made without departing from the scope of the invention as set forth in the claims.
This application claims the benefit of U.S. Provisional Application No. 62/875,235 filed Jul. 17, 2019 and 63/018,841 filed May 1, 2020, the entire contents of both of which are hereby fully incorporated herein by reference.
This invention was made with government support under Grant Nos. ECCS1710831, CBET1902503 and ECCS1912410, awarded by the National Science Foundation. The Government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind |
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PCT/US2020/042616 | 7/17/2020 | WO |
Number | Date | Country | |
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63018841 | May 2020 | US | |
62875235 | Jul 2019 | US |