Method and system for detecting reverse transcriptase activity by digital assay

Abstract

Methods and systems for detecting reverse transcriptase activity of a sample by digital assays. In an exemplary method, a reaction mixture comprising a reagent for reverse transcription, a sample, and an RNA polymer may be prepared. Complementary DNA (cDNA) may be synthesized in a reaction mixture using RNA polymers as templates. The amount of cDNA synthesized may be proportional to the reverse transcriptase activity of the sample. The partitions may be formed after synthesizing the cDNA. The partition may comprise a copy of the partially occupied cDNA. Each partition may contain a portion of the reaction mixture. A target representative of cDNA may be amplified in the partition. Amplification data of the target may be collected from the partitions.

Description

Cross-reference to prior applications

[0001] This application is based upon and claims the benefit of 35 U.S.C. §119(e) prior to U.S. Provisional Patent Application Serial No. 63 / 347,810, filed on June 1, 2022, which is hereby incorporated by reference in its entirety for all purposes. introduction

[0002] Reverse transcriptase is an enzyme that catalyzes the synthesis of complementary DNA from an RNA template, a process known as reverse transcription. Encoded by retroviruses, the enzyme's activity is essential for the retroviral life cycle, converting the viral RNA genome into DNA for integration into the host genome. The ability to reverse transcribe RNA into DNA has many practical applications and is required for a variety of laboratory protocols and clinical trials. Therefore, accurate, reliable, and safe quantitative testing of reverse transcriptase activity is important for quality control by enzyme manufacturers and for troubleshooting and analytical validation by end users.

[0003] Known methods for quantifying reverse transcriptase activity have various disadvantages. In one method, relative activity is measured by testing the reverse transcriptase preparation of interest with a "gold standard" reverse transcriptase. However, the activity of the gold standard may be unstable and may drift over time, making this method inaccurate and unreliable. In another method, the absolute activity of the reverse transcriptase preparation of interest is measured by quantifying the incorporation of radionucleotides into synthetic complementary DNA. Although measuring absolute activity is valuable, this radioactive method is undesirable due to the licensing, inspection, cost, training and hazards involved in the storage, handling and disposal of radioactive materials. Summary of the invention

[0004] The present disclosure provides methods and systems for detecting the reverse transcriptase activity of samples by digital experiments. In an exemplary method, a reaction mixture comprising a reagent, a sample and an RNA polymer for reverse transcription can be prepared. Complementary DNA (cDNA) can be synthesized in a reaction mixture using an RNA polymer as a template. The amount of the synthesized cDNA can be proportional to the reverse transcriptase activity of the sample. After synthesizing the cDNA, a partition can be formed. The partition can include copies of the cDNA occupied by a portion. Each partition can include a partial reaction mixture. A target representing a cDNA can be amplified in the partition. Amplification data of the target can be collected from the partition. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1 is a flow chart of exemplary steps that may be performed in a method for detecting reverse transcriptase activity in a sample by a digital assay.

[0006] Figure 2is a schematic flow chart showing the constructs produced during the performance of an exemplary method for detecting reverse transcriptase activity of a sample by a digital assay.

[0007] Figure 3 is shown in more detail Figure 2 Schematic diagram of aspects of an exemplary method.

[0008] Figure 4 is a diagram schematically showing how changing the initial concentration of RNA template affects the kinetics of reverse transcription product (cDNA) formation.

[0009] Figure 5 is a graph schematically illustrating the linear correlation between the concentration of reverse transcriptase [E] in a reverse transcription reaction and the resulting target copy number that can be quantified in a digital assay.

[0010] Figure 6 is a schematic diagram of an exemplary method for testing nucleic acids for reverse transcriptase activity in a sample by a digital assay.

[0011] Figure 7 is a scatter plot of amplification data of a target collected from a collection of four droplets formed from corresponding reaction mixtures with different amounts of reverse transcriptase activity to synthesize cDNA providing the target.

[0012] Figure 8 The target concentration detected in duplicate in the droplet pool is plotted against the reverse transcriptase concentration used to generate the cDNA providing the target in the bulk reaction mixture.

[0013] Fig. 9 is a plot of target concentrations detected in duplicate in a pool of droplets, generated in a bulk reaction mixture by reverse transcription of serial dilutions of each of two commercially available reverse transcriptases. DETAILED DESCRIPTION

[0014] Various aspects and examples of the method, system and composition for detecting the reverse transcriptase activity of a sample are described below and shown in the relevant drawings. Unless otherwise stated, methods, systems and compositions may (but not necessarily) include at least one of the structure, component, function and / or variation described, shown and / or included herein. In addition, unless specifically excluded, the processing steps, structures, components, functions and / or variations described, shown and / or included herein may be included in other similar methods, systems and compositions, including being interchangeable between disclosed examples. The description of various examples below is essentially merely illustrative and is by no means intended to limit examples, their applications or their uses. In addition, the advantages provided by the examples described below are essentially illustrative, and not all examples provide the same advantages or the same degree of advantages.

[0015] The present disclosure provides methods and systems for detecting the reverse transcriptase activity of a sample. In an exemplary method, a reaction mixture comprising a reagent, a sample and an RNA polymer for reverse transcription can be prepared. Complementary DNA (cDNA) can be synthesized in a reaction mixture using an RNA polymer as a template. The amount of the synthesized cDNA can be proportional to the reverse transcriptase activity of the sample. After synthesizing the cDNA, a partition can be formed. The partition can include copies of the cDNA occupied by a portion. Each partition can include a partial reaction mixture. A target representing the cDNA can be amplified in the partition. Amplification data of the target can be collected from the partition.

[0016] Other aspects of the disclosure are described in the following subsections: (I) Definitions, (II) Overview, (III) Examples, Components and Alternatives, (IV) Illustrative Combinations and Additional Examples, (V) Advantages and Benefits, and (VI) Conclusion.

[0017] The features, functions, and advantages can be achieved independently in various examples of the present disclosure or may be combined in yet other examples, further details of which can be seen with reference to the following description and drawings. I. definition

[0018] The technical terms used in the present disclosure have meanings recognized by those skilled in the art. However, the following terms may be further defined as follows.

[0019] "Amplicon" is the product of an amplification reaction. A copy of an amplicon can be generated by amplifying a target sequence so that the amplicon corresponds to (i.e., matches and / or is complementary to) the target sequence. However, the sequence of the amplicon, such as the sequence at the primer binding site, may not completely match and / or be complementary to the target sequence.

[0020] "Amplification" is a process of preparing multiple copies of an amplicon by matching, complementing and / or otherwise corresponding to a target sequence. The process is interchangeably referred to as an amplification reaction. As the amplification proceeds, the amplification may produce an exponential increase in the number of copies. Typical amplification may increase the number of copies of the amplicon by more than 100 times or 1,000 times. An exemplary amplification reaction of the method disclosed herein may include a polymerase chain reaction (PCR) or a ligase chain reaction (LCR), each of which is driven by a thermal cycle. The method may also use or alternatively use other amplification reactions, which may be carried out under isothermal conditions, such as branched probe DNA detection, cascade RCA, helicase-dependent amplification, loop-mediated isothermal amplification (LAMP), nucleic acid-based amplification (NASBA), nickase amplification reaction (NEAR), PAN-AC, Q-β replicase amplification, rolling circle replication (RCA), self-sustaining sequence replication, strand displacement amplification and / or similar means. Amplification may utilize a linear or circular template.

[0021] An "amplifier" is any reagent that promotes the generation of an amplicon by amplifying a target sequence. The reagent may include any combination of the following: at least one primer or primer pair for amplifying at least one target sequence, at least one marker for detecting the amplification of the at least one target sequence (e.g., at least one probe including a fluorophore and / or an intercalating dye as a marker), at least one polymerase and / or ligase (which may be thermostable), and nucleoside triphosphates (dNTPs and / or NTPs), etc.

[0022] A "carrier fluid" is a fluid that contacts the partitions, optionally surrounding the partitions. The fluid can be a liquid or a gas. The carrier fluid can be described as a continuous phase, and wherein the partitions are described as dispersed phases. The carrier fluid can be immiscible with the partitions and encapsulate the partitions. In some instances, the carrier fluid can be an oil, such as, for example, a fluorocarbon oil or a silicone oil.

[0023] "Complementary" means the rule of base pairing. If the first nucleic acid polymer or its region can hybridize with the second nucleic acid polymer in an antiparallel manner by forming a continuous (uninterrupted) or almost continuous base pair series (for example, at least 5, 6, 7, 8, 9 or 10 continuous base pairs), the first nucleic acid polymer or its region is "complementary" to the second nucleic acid polymer. If the hybridization of the first nucleic acid polymer (or its region) with the second nucleic acid polymer forms a continuous base pair series with each nucleotide in the first nucleic acid polymer or its region, the first nucleic acid polymer (or its region) is referred to as "completely complementary" to the second nucleic acid polymer. The "complement" of the first nucleic acid polymer or its region is the second nucleic acid polymer or its region that is completely complementary to the first nucleic acid polymer or its region. The "complementarity" between the first nucleic acid polymer (or its region) and the second nucleic acid polymer (or its region) refers to the number or percentage of base pairs that can be formed when the first nucleic acid polymer (or its region) is optimally arranged to hybridize with the second nucleic acid polymer (or its region) in an antiparallel manner. A first nucleic acid polymer or region thereof that is complementary to a second nucleic acid polymer or region thereof typically has a complementarity of at least 80%, 90%, 95% or 100%.

[0024] "Complementary DNA" (cDNA) is a DNA polymer that is complementary to a given RNA polymer, wherein the RNA polymer is used as a template for the synthesis of a DNA polymer, typically in the presence of a reverse transcriptase enzyme.

[0025] “Comprising,” “including,” and “having” (and variations thereof) are used interchangeably and mean including but not necessarily limited to, and are open-ended terms that are not intended to exclude additional, unrecited elements or method steps.

[0026] A "digital test" is one or more research procedures capable of detecting a single copy of an analyte (e.g., a nucleic acid target) in a collection of subsamples or partitions, wherein each subsample / partition of only a subset of the subsamples / partitions contains one or more copies of the analyte. A "digital amplification test" is a digital test that utilizes an amplification reaction to facilitate the detection of a single copy of a target. A digital test may be performed with any suitable number of subsamples / partitions that give a statistically significant result, such as at least twenty, one hundred, one thousand, or ten thousand, etc.

[0027] A "droplet" is a small volume of liquid surrounded by an immiscible liquid (e.g., encapsulated by an immiscible liquid that may form a continuous phase of an emulsion). The immiscible liquid may include oil and / or may consist primarily of oil. The droplets disclosed herein may, for example, have an average volume of less than about 500 nL, 100 nL, 10 nL, or 1 nL, etc.

[0028] An "enumerated value" is any value generated by enumerating a subset of target definitions for a set of subsamples / partitions in a digital assay. For example, an enumerated value may represent the number of subsamples / partitions in a set of subsamples / partitions that are positive for the presence of a given target or two or more given targets, negative for the presence of a given target or two or more given targets, positive only for a specified subset of one or more targets in a set of targets, negative only for a specified subset of one or more targets in a set of targets, etc.

[0029] “Exemplary” means “illustrative” or “serving as an example.” Likewise, the term “example” (or “exemplary”) means “illustrative.” Neither word implies desirability or superiority.

[0030] "First," "second," "alpha," "beta," and similar terms are used to distinguish or identify members of a group, etc., in the order in which they are introduced in a particular context, and are not intended to indicate an order or numerical limitation.

[0031] "Fluorescence" is optical radiation emitted in response to the absorption of light. As used herein, fluorescence is intended to encompass any form of photoluminescence in which the absorption of one or more photons promotes an electron to an excited state and results in the subsequent emission of new photons, whether from a singlet, triplet, or other state. The excited state resulting from the absorption may have any suitable lifetime.

[0032] "Fluorophore" refers to any atom, functional group, molecule or substance that is capable of fluorescing.

[0033] "Label" is any detectable marker or identifier associated with a member, structure or substance, such as any detectable marker or identifier associated with a primer, probe, amplicon, subsample, partition, etc. The marker can be covalently associated with a member, structure or substance (e.g., the marker is coupled to an oligonucleotide or antibody) or non-covalently associated (e.g., by embedding, hydrogen bonding, electrostatic interaction, encapsulation, etc.). Exemplary markers include optical markers, radioactive markers, magnetic markers, electrical markers, epitopes, enzymes, antibodies, oligonucleotides, etc. Optical markers can be optically detected by their interaction with light. Possibly suitable exemplary optical markers include fluorophores and quenchers, etc.

[0034] "Labeling" is any action, process or procedure that links or associates a label with a specified structure, member or substance. Labeling can result in non-covalent linking or association, or covalent attachment of the label to a structure, member or substance.

[0035] A "nucleic acid polymer" is a molecule or molecular duplex of any length comprising naturally occurring nucleotides (e.g., the polymer is an RNA polymer (also referred to as RNA) or a DNA polymer (also referred to as DNA)), or a synthetically produced compound that can hybridize with DNA or RNA in a sequence-specific manner similar to two naturally occurring nucleic acids, for example, can participate in Watson-Crick base pairing interactions. Nucleic acid polymers can contain any suitable number (such as at least about 5, 10, 100, or 1000, etc.) of nucleotides. The term "nucleic acid" refers to one or more nucleic acid polymers.

[0036] Nucleic acid polymers can have natural or artificial structures, or a combination of the two. Nucleic acid polymers with natural structures, i.e., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), generally have a backbone of alternating pentose groups and phosphate groups. Each pentose group is connected to a core base (e.g., purine (e.g., adenine (A) or guanine (T)) or a pyrimidine (e.g., cytosine (C), thymine (G) or uracil (U))). Nucleic acid polymers with artificial structures are analogs of natural nucleic acids, and can, for example, be produced by changing the pentose and / or phosphate groups and / or one or more core bases of the natural backbone. Exemplary artificial nucleic acid polymers include glycol nucleic acids (GNA), peptide nucleic acids (PNA), locked nucleic acids (LNA), threose nucleic acids (TNA), heterologous nucleic acids (XNA), etc.

[0037] The sequence of a nucleic acid polymer is determined by the order in which the nucleobases are arranged along the backbone. This order generally determines the ability of a nucleic acid polymer to hybridize with another nucleic acid through hydrogen bonding. Specifically, adenine pairs with thymine (or uracil), and guanine pairs with cytosine.

[0038] "Oligonucleotide" is a relatively short and / or chemically synthesized nucleic acid polymer. For example, the length of the oligonucleotide can be 5 to 1000 nucleotides, etc. In some cases, the oligonucleotide can be labeled with at least one marker, which can be coupled to any suitable structure of the oligonucleotide. The at least one marker can include at least one fluorophore, and therefore can be a fluorescent marker. Each marker can be coupled to the oligonucleotide at any suitable position, including 5' end, 3' end or middle 5' and 3' end.

[0039] "Optical radiation" means electromagnetic radiation in the spectrum, i.e. ultraviolet light, visible light and / or infrared light. Thus, the term "light" and optical radiation have the same meaning.

[0040] "Partitions" are discrete volumes of fluid (i.e., fluid volumes) that are spatially separated from one another (also referred to as separation volumes). Each partition in a set of partitions may contain portions of the same sample. Partitions may be separated from one another by fluids (e.g., oil or air), walls of a device, or a combination thereof, etc. Thus, partitions may be, for example, droplets of an emulsion, or volumes held by separate structures that hold fluids, such as wells (e.g., microwells each having a capacity of less than 1 μL), chambers (e.g., microchambers each having a capacity of less than 1 μL), tubes (e.g., microtubes each having a diameter of less than 1 mm), or microfluidic devices, etc. Partitions may have the same diameter as one another and / or may contain the same amount of fluid as one another.

[0041] "Partially occupied" means present or contained in only a subset of a set of partitions. A set of partitions containing a partially occupied target means that at least one copy of the target is present in each partition of only a subset of the partitions. In other words, one or more partitions do not contain any copies of the target. Copies of the target may be randomly distributed between partitions, but the supply may be limited so that no partition can receive a copy of the target.

[0042] A "partition count" (or "subsample count") is a value for the number of partitions (or subsamples) in a specified group, e.g., a population of partitions (or subsamples) having a specified target content. A partition count or subsample count may be an enumerated value. Partition / subsample counts may be determined by counting, estimating, and / or calculating.

[0043] The term "positive" when used to indicate the target content of a partition, subsample, or a population of partitions / subsamples indicates that each partition / subsample of a population contains (or at least appears and / or is considered to contain) at least one copy of a given target or each target of a given target set. The term "negative" when used to indicate the target content of a partition / subsample or a population of partitions / subsamples indicates that each partition / subsample of a population does not contain (or at least appears and / or is considered not to contain) at least one copy of each target of a given target or a given set of two or more targets.

[0044] "Primer" is an oligonucleotide (DNA / RNA or its analogue) that can serve as a starting point for template-directed nucleic acid synthesis or ligation under appropriate reaction conditions (e.g., in the presence of a template to which the primers are annealed, a nucleoside triphosphate, and a polymerizing agent (such as a DNA or RNA polymerase or ligase, or a reverse transcriptase), in an appropriate buffer and at an appropriate temperature). Primers can have any suitable length, such as 5 to 500 nucleotides, etc. The primers can be members of a "primer pair" or "primer set", which includes a "forward primer" and a "reverse primer", which define the ends of the amplicon produced in the amplification reaction. (The adjectives "forward" and "reverse" are arbitrarily specified relative to each other). The forward primer hybridizes with a complementary sequence to the 5' end region of the template sequence to be amplified, and the reverse primer hybridizes with a 3' end region of the template sequence. The term "primer binding site" refers to the portion of the template (or its complement) to which the primers are annealed. The full-length sequence of the primer does not need to be completely complementary to the primer binding site, as long as there is sufficient complementarity under the reaction conditions to anneal. Thus, a primer may have a 3' terminal region that is complementary to the primer binding site and a 5' terminal region that is not complementary to the primer binding site (and forms a "5'-tail").

[0045] A "probe" is any substance configured to facilitate or enable testing, identification and / or detection of an analyte. For example, a probe can be an amplification probe, which can include an oligonucleotide. The amplification probe is configured to detect the occurrence of an amplification reaction and / or the amplicon formed by the amplification reaction. In some instances, the amplification probe can be a fluorescent probe comprising an oligonucleotide labeled with a fluorophore. The amplification probe can be configured to hybridize with at least a portion of the amplicon produced by amplification. For example, the amplification probe can be a hydrolysis probe, a molecular beacon probe, a strand displacement probe, or a labeled primer, etc.

[0046] "Reverse transcriptase" is an enzyme that catalyzes "reverse transcription," which is the synthesis of complementary DNA (cDNA) using an existing RNA polymer as a template. cDNA can be synthesized by extending a primer that hybridizes to the RNA polymer. This extension can utilize one or more deoxyribonucleoside triphosphates (dNTPs) as substrates for primer extension.

[0047] "Reverse transcriptase activity" is the ability of a sample, stock solution, and / or enzyme preparation containing reverse transcriptase to catalyze reverse transcription. Any suitable activity unit can be used to define this activity. For example, a unit can be defined as the amount of enzyme required to catalyze the synthesis of a specified amount of cDNA, incorporate a specified amount of nucleotides into cDNA, etc., under specified reaction conditions (e.g., temperature, reaction duration, one or more RNA template sequences and concentrations, one or more primer sequences and concentrations, one or more dNTP concentrations, buffer composition, etc.). Activity can be defined as per unit volume, per enzyme mass, per enzyme number / mole, per total protein mass, etc.

[0048] A "subsample" is a smaller sample provided by a bulk sample or sample-containing fluid, containing only a portion of the bulk sample or sample-containing fluid. A collection of subsamples may be a collection of partitions, and vice versa.

[0049] "Substantially" means conforming primarily to the particular size, range, shape, concept or other aspect modified by the term, such that the feature or component does not need to conform exactly as long as it is suitable for its intended purpose or function. For example, a "substantially cylindrical" object means that the object resembles a cylinder, but may have one or more deviations from a true cylinder.

[0050] "Target / target" (also referred to as "target sequence / target sequence") is a nucleic acid polymer sequence (DNA and / or RNA) of any suitable length to be amplified in an amplification reaction. Exemplary target sequences are about 20-1000 nucleotides, or about 30-500 nucleotides, etc.

[0051] A "target-defined subset" of a set of partitions contains partitions with defined target content (positive / negative) for one or more targets.

[0052] A "template" is a nucleic acid polymer (eg, an RNA or DNA polymer) that serves as a model for the production of another nucleic acid polymer. II. Overview

[0053] This section provides an overview of the methods and systems described herein.

[0054] A method for detecting the reverse transcriptase activity of a sample is provided. In the method, a reaction mixture comprising a reagent for reverse transcription, a sample and an RNA polymer can be prepared. Complementary DNA (cDNA) can be synthesized in the reaction mixture using the RNA polymer as a template. The amount of the synthesized cDNA can be proportional to the reverse transcriptase activity of the sample. After synthesizing the cDNA, partitions can be formed. The partitions can include copies of the partially occupied cDNA. Each partition can include a partial reaction mixture. A target representing the cDNA can be amplified in the partitions. Amplification data of the target can be collected from the partitions.

[0055] A system for measuring the reverse transcriptase activity of a sample is provided. The system may include multiple partitions, the partitions comprising partially occupied cDNAs. Each partition may include a portion of a reaction mixture, wherein the cDNA is synthesized by reverse transcription using the reverse transcriptase activity of the sample and an RNA polymer as a template. The cDNA may be present in an amount proportional to the reverse transcriptase activity of the sample. If a copy of the cDNA is present in the partition, each partition may also include a reagent sufficient to amplify a target representing the cDNA. III. Examples, components and alternatives

[0056] Subsections A to D below describe selected aspects of an exemplary method and system for detecting reverse transcriptase activity of a sample by digital testing. The embodiments of these subsections are intended to illustrate and should not be construed as limiting the full scope of the disclosure. Each subsection may include one or more different embodiments and / or context or related information, function, structure and / or process. A. Method for detecting reverse transcriptase activity

[0057] This subsection describes an illustrative method for using a digital assay to detect reverse transcriptase assays; see Figure 1 Each method can include Figure 1 Any suitable combination of the steps listed in flowchart 100, performed in any suitable order, and any step omitted or performed any suitable number of times, and using or including any features, aspects, reagents, configurations, additional steps or modifications described elsewhere in the present disclosure.

[0058] In the selection step 102, a sample containing reverse transcriptase activity is selected. The sample may contain a reverse transcriptase that has been purified, expressed by an engineered construct, modified to change its primary amino acid sequence relative to a native reverse transcriptase (e.g., to eliminate RNase H activity, increase thermal stability, change fidelity, etc.), or any combination thereof. In other cases, the sample may contain a reverse transcriptase that has been expressed in one or more infected cells.

[0059] In preparation step 104, a reaction mixture including a sample, an RNA polymer, at least one reverse transcription (RT) primer and a reverse transcription reagent can be prepared. The RNA polymer may have a predefined sequence or may include two or more RNA molecules with different sequences. The RNA polymer may be expressed by an engineered construct and / or may be purified. Each RT primer may independently have a single nucleotide sequence or may be a degenerate primer. The RT primer may be complementary to the RNA polymer and configured to hybridize with the RNA polymer, with the position being separated from the 5' end of the RNA polymer (e.g., closer to the 3' end of the RNA polymer than the 5' end). Depending on the nucleotide requirements of the cDNA to be synthesized, the reagent may include one or more dNTPs (e.g., dATP, dCTP, dGTP and / or dTTP).

[0060] Reaction mixture can also be prepared at this stage to include all amplification reagents that are enough for subsequent target amplification.Therefore, reaction mixture can include at least one amplification primer or at least one pair of amplification primers for target amplification, and the enzyme for catalytic amplification.Enzyme can be an archaeal dna polymerase (for example, a thermostable polymerase) for extending one or more amplification primers, or a DNA ligase (for example, a thermostable DNA ligase) for connecting amplification primers to each other.In other embodiments, one or more amplification reagents can be added to the reaction mixture after reverse transcription.

[0061] In the synthesis step 106, complementary DNA (cDNA) can be synthesized in the reaction mixture using RNA polymer as a template. The synthesis step 106 can be performed at one or more controlled temperatures and can be started and stopped by changing the temperature of the reaction mixture. For example, the synthesis can be started by heating the reaction mixture to a first predetermined temperature, and the synthesis can be stopped by heating or cooling the reaction mixture to a second predetermined temperature. In other embodiments, the synthesis step 106 can be started by adding one or more reagents for reverse transcription, and / or the synthesis step 106 can be stopped by adding a reverse transcription inhibitor.

[0062] In forming step 108, partitions comprising partially occupied cDNA can be formed. Each partition comprises a portion of the reaction mixture. Only each partition of a subset of partitions comprises at least one cDNA copy. Forming step 108 may include dividing the reaction mixture into spatially separated fluid volumes. Spatially separated fluid volumes may be separated from each other by immiscible fluids (e.g., when the volumes are droplets or clumps), or each volume may be accommodated by a separate fluid holding structure (e.g., a hole (e.g., micropore), a chamber (e.g., a microchamber), etc.).

[0063] In the amplification step 110, a target representing a cDNA can be amplified in the partition. The target can be provided by the cDNA and can correspond to any suitable portion of the cDNA. The amplification step 110 can include thermal cycling the partition through multiple cycles. Each cycle can include a denaturation phase, an annealing phase, and an extension phase, which can be achieved by heating the partition to a denaturation temperature, cooling the partition to an annealing temperature, and heating the partition to an extension / ligation temperature. In other embodiments, the annealing temperature and the extension / ligation temperature can be the same.

[0064] In the collecting step 112, amplification data of the target can be collected from the partitions. The collecting step 112 can be performed after the amplification step 110 is completed. The amplification data can be collected by detecting light (e.g., fluorescence) from the partitions. The fluorescence intensity detected from each partition can depend on whether the target is amplified in the partition.

[0065] In the enumeration step 114, at least one population of the partitions can be enumerated, wherein at least one population is defined using amplification data. The signal detected from each partition in the collection step can be compared with a threshold value to classify the partition as target positive or negative. Positive or negative partition counts for partitions where the target is negative or the target is positive can be obtained. Total partition counts can also be obtained. Positive or negative partition counts and total partition counts can be used to calculate the score of the partition where the target is negative or the target is positive. The average target concentration of each partition can be calculated based on the score, for example, using Poisson statistics. The target concentration per unit volume can be calculated based on the average volume or total volume of the partition.

[0066] In the determination step 116, the value of the reverse transcriptase activity of the sample can be determined based on the enumeration. Since each target represents a cDNA molecule, the target concentration obtained above can be used to calculate the amount of cDNA synthesized in the reverse transcription reaction by the amount of reverse transcriptase present in the sample. Since there is a linear correlation between the amount of enzyme and the amount of target / cDNA synthesized, the amount of target / DNA synthesized can be used to determine the absolute enzyme activity without reference to the reverse transcriptase. B. Illustrative Flowchart

[0067] This subsection describes a schematic flow chart 220 showing the configurations generated during the execution of an exemplary method for detecting reverse transcriptase activity in a sample 222 by a digital assay; see Figure 2 and 3 Further exemplary aspects of the method are described in Subsection A above.

[0068] Sample 222 can be combined with RNA polymerase 224 and reagents 226 for reverse transcription (RT) to prepare reaction mixture 228 (see Figure 2). The components of reaction mixture 228 can be combined with each other in any suitable order. For example, RNA polymer and RT reagent can be present together in a preformed composition and combined with the sample.

[0069] Sample 222 may include at least one copy of reverse transcriptase 230. The sample may be a commercially available / laboratory sample that includes commercially produced and / or engineered reverse transcriptase, which may be expressed and / or purified from an engineered construct. In other cases, the sample may be a clinical sample, an environmental sample, etc., which may or may not include a naturally expressed reverse transcriptase.

[0070] RNA polymer 224 can be composed primarily or entirely of copies of the same RNA sequence. RNA polymers can be expressed from engineered constructs and / or can be purified.

[0071] RT reagent 226 may include at least one copy of RT primer 232 and dNTP 234 (deoxyribonucleoside triphosphates) for reverse transcriptase 230 to catalyze extension of the RT primer. Each RT primer 232 is configured to anneal to RNA polymer 224 through base pairing, so that extension of the primer uses the RNA polymer as a template.

[0072] The reaction mixture 228 contains a significant excess of RNA polymer 224 copies compared to the reverse transcriptase 230 copies. The excess of RNA polymer 224 (and RT reagent 226) ensures that the reverse transcriptase remains rate-limited for reverse transcription throughout the duration of the reaction until the reaction is stopped. When the reaction mixture 228 is first formed, the molar ratio of RNA polymer 224 to reverse transcriptase 230 can be at least 10:1, 50:1, 100:1, 500:1, or 1000:1, etc. RT primer 232 can be present in equal amounts or in molar excess to RNA polymer 224, as shown.

[0073] The reaction mixture 228 is shown on the left as an initial, unreacted configuration 236 and as a final, reacted configuration 238 after the reverse transcription reaction has been stopped. Incubating the reaction mixture at one or more defined temperatures for a defined time interval allows reverse transcriptase 230 to catalyze the synthesis of cDNA 240 by extending RT primer 232 using RNA polymer 224 as a template. In this highly simplified example, each copy of reverse transcriptase 230 catalyzes only three cycles of cDNA synthesis, but in other examples, each active copy of reverse transcriptase 230 may be responsible for synthesizing an average of at least 10, 50, 100, 500, or 1000 copies of cDNA 240. The cDNA may be single-stranded, as shown, or may hybridize with RNA in the form of an RNA / DNA hybrid or intramolecularly hybridize with itself to create a stem-loop structure, etc.

[0074] The reverse transcription reaction can be stopped at the end of the specified time interval by inactivating the reverse transcriptase 230 (indicated by an "X" in each copy of the reverse transcriptase). In some examples, inactivation can be performed by heating the reaction mixture to an inactivation temperature that is higher than the specified reaction temperature to denature the reverse transcriptase. The inactivation temperature can be, for example, at least 60, 70, or 80 degrees.

[0075] Partitions 242 can be formed, each containing a portion of the reaction mixture 228. The partitions contain partially occupied cDNA 240, so that only a subset of the partitions 242 contain copies of the cDNA, as shown. Each partition can contain the same volume of reaction mixture. Each partition also contains reagents sufficient to amplify the target 244 from the cDNA 240 in the partition 242, as shown by the amplification arrow 246, to generate an amplicon 248.

[0076] Amplification data 250 can be collected from the partitions 242, as shown in a graph 252 of the signal amplitude (e.g., fluorescence intensity) detected from each individual partition 242. In this example, negative partitions 254 (no target amplification) have weaker signals, while positive partitions 256 with target amplification have stronger signals. The signal from each partition can be compared to a threshold 258 to designate the partition as negative or positive.

[0077] Amplification data can be processed, as shown in processing arrow 260, to determine the reverse transcriptase activity of the sample. For example, negative partition 254 or positive partition 256 and total partition 242 (positive and negative) can be enumerated to obtain enumeration value (partition count). Reverse transcriptase activity can be defined according to any suitable convention. For example, one or more enumeration values ​​can be utilized to calculate the average value of the target copy (and / or cDNA copy) present in each partition and / or unit volume, and / or the total value of the target copy (and / or cDNA copy) present in the reaction mixture or in the reaction mixture or sample per unit volume.

[0078] Figure 3 Shows in more detail Figure 2Further illustrative aspects of an exemplary method of. A hybrid 262 of a copy of an RT primer 232 annealed to a copy of an RNA polymer 224 is shown at the top. A copy of cDNA 240 generated by extension of RT primer 232 is shown below hybrid 262. Amplification primer pairs 264a, 264b and hydrolysis probe 266 are shown positioned along the cDNA copy, each hybridizing with the cDNA (or its complement) at the location. A copy of amplicon 248 generated by amplifying target 244 with primers 264a, 264b, and a degraded form of hydrolysis probe 266 are shown below the primers. The hydrolysis probe includes a fluorophore 268 and a quencher 270. Due to the degradation of the hydrolysis probe caused by target amplification, the fluorescence of the fluorophore 268 is increased because the quencher 270 is no longer tethered to the vicinity of the fluorophore. C Exemplary kinetic relationships in reverse transcription experiments

[0079] This section describes the kinetic relationship between the concentrations of substrate (RNA polymer), product (cDNA synthesized using RNA polymer as template), and enzyme (reverse transcriptase) in a reverse transcription assay; see Figure 4 and Figure 5 , they are schematic in nature and do not provide actual data.

[0080] Figure 4 It illustrates how changing the initial concentration of RNA polymer (template) may affect the kinetics of product (cDNA) formation catalyzed by reverse transcriptase while keeping the reverse transcriptase concentration constant. Three reverse transcription reactions performed for increasing initial concentrations of RNA polymer ([RNA]1, [RNA]2, and [RNA]3) give product accumulation curves 372a, 372b, and 372c, respectively. The initial rate V0 of each reaction is represented by the slope of the corresponding rate line 374a, 374b, and 374c that is tangent to the corresponding accumulation curve and intersects the origin. At the two lower concentrations of RNA polymer [RNA]1 and [RNA]2, the enzyme is substrate unsaturated at the beginning of the reaction, so V0 is less than the maximum rate V0. max , and product accumulation curves 372a and 372b diverge from rate lines 374a and 374b early in the reaction. In contrast, at the highest concentration of RNA polymer [RNA]3, the enzyme is substrate saturated at the beginning of the reaction and remains substrate saturated during the linear phase of the reaction, producing a linear portion 376 of accumulation curve 372c. During the linear phase, V0 is equal to V max The reverse transcription (RT) reactions disclosed herein can be performed with a concentration of RNA polymers sufficient to saturate the reverse transcriptase with RNA polymers from the beginning to the end of each RT reaction. Thus, the RT reactions are maintained at V max , the product accumulated linearly during the entire reaction period.

[0081] Formula 1 defines V max The total concentration of active enzyme [E t ] (i.e., the number of active enzyme sites per unit volume): V max =k cat [E t ] (1) where k cat is the catalytic constant or turnover number, which is a measure of the number of substrate molecules converted to product per unit time (e.g., per second) per enzyme active site. cat For a given enzyme and set of reaction conditions (i.e. temperature, buffer, pH, etc.) is constant, so V max is proportional to the total concentration of active enzyme under these reaction conditions. Therefore, V max The measured value of can be used as a surrogate for the concentration of active reverse transcriptase, regardless of k cat Whether the value is known.

[0082] Figure 5 A graph illustrating the linear relationship between the concentration of reverse transcriptase present in the RT reaction (E) and the number of copies of the cDNA product synthesized per unit volume and per unit time (P) in the RT reaction is shown. Each RT reaction is represented by a triangle in the graph. The number of cDNA copies per unit volume and time is equal to the V of the RT reaction. max , and can be measured in digital assays by amplification of targets present in cDNA. D. Example test results

[0083] This section describes exemplary test results obtained by using a digital assay to detect reverse transcription activity; see Figure 6-9 .

[0084] Figure 6 The schematic diagram of the nucleic acid polymer constructed or synthesized is shown. A 1.5 kb expression construct 478 is amplified from a plasmid, purified and analyzed on a Bioanalyzer High Sensitivity DNA chip (Agilent Technologies). The expression construct 478 has a T7 RNA polymerase promoter sequence 480 fused to an artificial ("external") template sequence 482. The artificial template sequence is transcribed in vitro by T7 RNA polymerase into a 1.5 kb foreign RNA polymer 424, which is then purified by DNA enzyme I digestion and lithium chloride precipitation.

[0085] The RT reaction including RNA polymerase 424 as a template was performed as follows. The RT reaction mixture was prepared on ice in a 96-well PCR plate. Each reaction mixture contained 1x RT buffer (50 mMTris pH 8.3, 75 mM KCl, 3 mM MgCl2); 0.5 U / μL RNase inhibitor (mouse, New England Biolabs); RT primers 432a and 432b, each at 1 μM; dNTPs, each at 1 mM; RNA polymerase 424 at a concentration of 50 nM; iScript TM Advanced reverse transcriptase (Bio-Rad) was used at a concentration of 0.8-1.4 nM. The PCR plate was transferred to a thermal cycler and incubated at 4°C for 30 minutes, 39°C for 20 minutes, 85°C for 10 minutes, and maintained at 4°C. cDNA 440a, 440b was synthesized by extending the corresponding RT primers 432a, 432b. Each RT reaction was measured in a collection of droplets by digital PCR using amplification primer pairs 464a, 464b and a fluorescent probe to amplify the target 444 present in the cDNA 440a, 440b.

[0086] Figure 7 A scatter plot of amplification data collected from a collection of four droplets (AD) containing cDNA 440a, 440b synthesized with different and increasing concentrations of reverse transcriptase (see also Figure 6 ). Droplets (440a and / or 440b) deemed cDNA-positive have a fluorescence amplitude above a threshold value 458, while droplets deemed cDNA-negative have a fluorescence amplitude below the threshold value.

[0087] Figure 8 The concentration (copies / μL) of target 444 measured from a pool of replicate RT reactions is shown along with the iScript used to generate cDNA 440a, 440b in each pool of replicate RT reactions. TM The concentration of advanced reverse transcriptase (see also Figure 6 ). Error bars represent standard deviations of replicates per set. The coefficient of determination (R2) was 0.992 and the coefficient of variation was less than 10%. The assay can detect a 20% difference in reverse transcriptase activity / concentration.

[0088] Fig. 9A graph is shown plotting the concentration of a target 444 amplified from cDNA 440a, 440b synthesized as described above, except that two other commercially available reverse transcriptases were used to catalyze the reverse transcription reaction (see also Figure 6 ). Sample set AF was a serial two-fold dilution of Reliance RT (Bio-Rad), and sample set GN was a serial two-fold dilution of RTX (New England Biolabs). IV. Illustrative Combinations and Additional Embodiments

[0089] This section describes additional aspects and features of the methods and systems of the present disclosure, presented as, but not limited to, a series of paragraphs, some or all of which may be alphanumerically indexed for clarity and efficiency. Each of these paragraphs may be combined in any suitable manner with one or more other paragraphs, and / or with the disclosure elsewhere in this application. Some of the paragraphs below explicitly mention and further limit other paragraphs, providing examples of some suitable combinations, but are not limited thereto.

[0090] A1. A method for detecting reverse transcriptase activity of a sample, the method comprising: preparing a reaction mixture, the reaction mixture comprising a reagent for reverse transcription, a sample and an RNA polymer; in the reaction mixture, synthesizing complementary DNA (cDNA) using the RNA polymer as a template, wherein the amount of cDNA synthesized is proportional to the reverse transcriptase activity of the sample; forming partitions after synthesis, the partitions comprising partially occupied copies of the cDNA, each partition comprising a portion of the reaction mixture; amplifying targets representing the cDNA in the partitions; and collecting amplification data of the targets from the partitions.

[0091] A2. The method of paragraph A1 further comprises enumerating at least one population of partitions defined using amplification data based on positivity or negativity of the target.

[0092] A3. The method as described in paragraph A2 also includes enumerating the partitions to obtain a total number of partitions regardless of the positivity / negativity of the target.

[0093] A4. The method of paragraph A2 or A3, further comprising determining a value of reverse transcriptase activity based on the enumeration.

[0094] A5. The method of paragraph A4, wherein determining comprises calculating a fraction of partitions that are target positive or a fraction of partitions that are target negative.

[0095] A6. A method as described in paragraph A4 or paragraph A5, wherein determining includes calculating the average number of cDNA molecules per partition or unit volume.

[0096] A7. The method of any of paragraphs A1-A6, wherein preparing the reaction mixture comprises combining the sample and the RNA polymer with each other.

[0097] A8. The method of any of paragraphs A1-A7, wherein the RNA polymer is a product of in vitro transcription.

[0098] A9. A method as described in any of paragraphs A1-A8, wherein the sample includes at least one reverse transcriptase that produces reverse transcriptase activity, and wherein after preparing the reaction mixture, the RNA polymer is present in at least a 20-fold, 50-fold, or 100-fold molar excess relative to the at least one reverse transcriptase.

[0099] A10. The method of any one of paragraphs A1-A9, wherein the reagents for reverse transcription include deoxyribonucleoside triphosphates (dNTPs) and at least one primer for cDNA synthesis using RNA polymers as templates.

[0100] A11. The method of any of paragraphs A1-A10, wherein the sample comprises at least one reverse transcriptase that produces reverse transcriptase activity, and wherein the RNA polymer is present at a non-limiting concentration relative to the at least one reverse transcriptase throughout the synthesis process.

[0101] A12. The method of any of paragraphs A1-A11, wherein the cDNA accumulates in the reaction mixture at a substantially constant rate during synthesis.

[0102] A13. The method of any of paragraphs A1-A12, wherein the synthesizing comprises incubating the reaction mixture at a first temperature for a predetermined time interval and then heating the reaction mixture to a second temperature at the end of the predetermined time interval to stop synthesizing cDNA.

[0103] A14. The method of paragraph A13, wherein the first temperature is greater than 25 degrees Celsius and the second temperature is greater than 70 degrees Celsius.

[0104] A15. The method of paragraph A14, wherein the first temperature is at least 35 degrees Celsius and the second temperature is at least 75 degrees Celsius.

[0105] A16. The method of any of paragraphs A1-A15, wherein forming comprises forming partitions such that each partition comprises a pair of primers to amplify the target.

[0106] A17. The method of any of paragraphs A1 to A16, wherein amplifying comprises thermal cycling the partitions.

[0107] A18. A method as described in paragraph A17, wherein amplifying includes performing a polymerase chain reaction in the partitions.

[0108] A19. The method of any of paragraphs A1 to A18, wherein collecting amplification data comprises detecting fluorescence from the partitions.

[0109] A20. The method of paragraph A19, wherein forming comprises forming the partitions so that each partition contains a probe having a label, wherein the probe specifically binds to the target, and wherein detecting fluorescence comprises detecting fluorescence from the label.

[0110] A21. The method of any of paragraphs A1-A20, wherein forming partitions comprises forming droplets.

[0111] A22. The method of any of paragraphs A1-A21, wherein the reverse transcriptase activity of the sample determines the accumulation rate of cDNA during the entire synthesis process.

[0112] A23. A system for measuring the reverse transcriptase activity of a sample, the system comprising: a plurality of partitions containing partially occupied cDNA, each partition containing a portion of a reaction mixture, wherein the cDNA is synthesized by reverse transcription using the reverse transcriptase activity of the sample and RNA polymers as a template, if a copy of the cDNA is present in the partition, the cDNA is present in an amount proportional to the reverse transcriptase activity of the sample, and each partition also contains reagents sufficient to amplify a target representing the cDNA.

[0113] A24. The system of paragraph A23, further comprising a retainer for accommodating the plurality of partitions. V. Advantages and Benefits

[0114] The various embodiments of the methods and systems for detecting reverse transcriptase activity provide several advantages over known solutions for detecting reverse transcriptase activity. For example, the illustrative examples described herein allow for measuring reverse transcriptase activity without a reverse transcriptase standard, thereby avoiding inaccuracies and errors caused by activity drift that may occur in such a standard.

[0115] Furthermore, among other benefits, the illustrative examples disclosed herein allow for quantification of reverse transcriptase activity of a sample without the use of radioactive dNTP substrates.

[0116] Furthermore, among other benefits, the illustrative examples disclosed herein enable absolute quantification of reverse transcriptase activity.

[0117] Furthermore, among other benefits, the illustrative examples disclosed herein allow manufacturers of reverse transcriptases to measure their activity.

[0118] Furthermore, among other benefits, the illustrative examples disclosed herein allow an end user to measure the activity of a reverse transcriptase to ensure that the reverse transcriptase has a desired, sufficient, or specified level of activity.

[0119] In addition, among other benefits, the illustrative examples disclosed herein can be used to detect reverse transcriptase activity in cell culture supernatants or other biological samples. In some cases, the methods and systems can be used to detect and / or quantify retroviruses in cell supernatants or other biological samples.

[0120] There is no known method or system that is capable of performing these functions.However, not all embodiments described herein provide the same advantages or the same degree of advantages. VI. in conclusion

[0121] The above disclosure may include multiple different examples with independent utility. Although various of these disclosures have been disclosed in their preferred forms, the specific examples of the invention disclosed and illustrated herein should not be considered restrictive, because multiple changes can be made. With respect to the section titles used in this disclosure, such titles are only used for organizational purposes. The subject matter of the present disclosure includes all novel and non-obvious combinations and sub-combinations of the various elements, features, functions, processes and / or characteristics disclosed herein. The claims specifically point out certain combinations and sub-combinations that are novel and non-obvious. Other combinations and sub-combinations of elements, features, functions, processes and / or characteristics may be claimed in applications claiming priority to this application or a related application. These claims, whether their scope is wider, narrower, the same or different than the scope of the original claims, are also deemed to be included in the subject matter of the present disclosure.

Claims

1. A method for detecting reverse transcriptase activity of a sample, the method comprising: preparing a reaction mixture comprising reagents for reverse transcription, a sample, and an RNA polymerase; In the reaction mixture, complementary DNA (cDNA) is synthesized using the RNA polymer as a template, wherein the amount of cDNA synthesized is proportional to the reverse transcriptase activity of the sample; forming partitions after synthesis, the partitions comprising partially occupied copies of the cDNA, each partition comprising a portion of the reaction mixture; amplifying the target representing the cDNA in the partition; as well as Amplification data for the target is collected from the partitions.

2. The method of claim 1 further comprises enumerating at least one population of partitions defined using amplification data according to positivity or negativity of the target.

3. The method of claim 2, further comprising enumerating the partitions to obtain a total number of partitions regardless of positivity / negativity of the target.

4. The method of claim 2, further comprising determining a value of reverse transcriptase activity of the sample based on the enumeration. 5 . The method of claim 4 , wherein determining comprises calculating a fraction of partitions that are target positive or a fraction of partitions that are target negative.

6. The method of claim 4, wherein determining comprises calculating an average number of cDNA molecules per partition or unit volume.

7. The method of claim 1, wherein preparing a reaction mixture comprises combining the sample and the RNA polymer with each other.

8. The method of claim 1, wherein the RNA polymer is a product of in vitro transcription.

9. The method of claim 1, wherein the sample comprises at least one reverse transcriptase that produces reverse transcriptase activity, and wherein after preparing the reaction mixture, the RNA polymer is present in at least a 20-fold, 50-fold, or 100-fold molar excess relative to the at least one reverse transcriptase.

10. The method of claim 1, wherein the reagents for reverse transcription include deoxyribonucleoside triphosphates (dNTPs) and at least one primer for cDNA synthesis using RNA polymers as templates.

11. The method of claim 1, wherein the sample comprises at least one reverse transcriptase that produces reverse transcriptase activity, and wherein the RNA polymer is present at a non-limiting concentration relative to the at least one reverse transcriptase throughout the synthesis process.

12. The method of claim 1, wherein the cDNA accumulates in the reaction mixture at a substantially constant rate during synthesis.

13. The method of claim 1, wherein synthesizing comprises incubating the reaction mixture at a first temperature for a predetermined time interval and then heating the reaction mixture to a second temperature at the end of the predetermined time interval to stop synthesizing cDNA.

14. The method of claim 13, wherein the first temperature is above 25 degrees Celsius and the second temperature is above 70 degrees Celsius.

15. The method of claim 14, wherein the first temperature is at least 35 degrees Celsius and the second temperature is at least 75 degrees Celsius.

16. The method of claim 1, wherein forming comprises forming partitions such that each partition contains a pair of primers to amplify the target.

17. The method of claim 1, wherein amplifying comprises thermal cycling the partitions.

18. The method of claim 17, wherein amplifying comprises performing polymerase chain reaction in the partitions.

19. The method of claim 1, wherein collecting amplification data comprises detecting fluorescence from the partitions.

20. The method of claim 19, wherein forming comprises forming the partitions such that each partition contains a probe having a label, wherein the probe specifically binds to the target, and wherein detecting fluorescence comprises detecting fluorescence from the label.

21. The method of claim 1, wherein forming the partitions comprises forming droplets.

22. The method of claim 1, wherein the reverse transcriptase activity of the sample determines the accumulation rate of cDNA during the entire synthesis process.

23. A system for measuring reverse transcriptase activity of a sample, the system comprising: A plurality of partitions comprising partially occupied cDNA, each partition comprising a portion of a reaction mixture wherein the cDNA is synthesized by reverse transcription using reverse transcriptase activity of the sample and RNA polymerase as a template, if a copy of the cDNA is present in the partition, the cDNA is present in an amount proportional to the reverse transcriptase activity of the sample, and each partition further comprising reagents sufficient to amplify a target representing the cDNA.

24. The system of claim 23, further comprising a holder for accommodating the plurality of partitions.

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