Rapid reverse transcription quantitative polymerase chain reaction

Abstract

Provided herein is a method for rapidly detecting RNA in a sample, the method comprising: providing a reaction mixture containing the sample, an amplification reagent, and a polymerase enzyme having both RNA- and DNA-dependent polymerase activity; reverse transcribing the RNA to DNA by incubating for a reverse transcription time of 5 minutes or less; and amplifying the DNA by performing a thermal cycling protocol including multiple amplification cycles, each amplification cycle including at least a thermal denaturation step and an annealing step.

Description

Detailed Description of the Invention

[0001] [Technical field] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 793,657, filed January 17, 2019, which is hereby incorporated by reference in its entirety.

[0002] Provided herein are methods for rapidly amplifying and detecting RNA in a sample. In certain embodiments, the disclosed methods can be used in clinical diagnostics, such as detecting viral infections in subjects.

[0003] [Background technology] Infectious diseases are often diagnosed by nucleic acid testing. However, the most widely implemented methods for amplifying nucleic acids, polymerase chain reaction (PCR) or quantitative PCR or real-time PCR (qPCR or RT-PCR), are time- and energy-intensive. Although RNA detection can also be essential for clinical diagnosis, especially for viral infections, RNA cannot be directly amplified by PCR. RNA requires a first step of reverse transcription, in which an enzyme called reverse transcriptase enzymatically creates a DNA copy (cDNA) from the RNA template. In RT-qPCR, this cDNA is then amplified in a PCR reaction.

[0004] RT-qPCR assays can be performed in either one-step or two-step reactions. In one-step RT-qPCR, cDNA synthesis and qPCR are performed in a common reaction buffer in a single reaction vessel. In two-step RT-qPCR, cDNA is synthesized in one reaction and then an aliquot of cDNA is used for the subsequent qPCR experiment. One-step reactions allow for minimal sample handling and closed-tube reactions, reducing the chance of pipetting errors and cross-contamination. However, for single-tube RT-qPCR assays, the mixed RT and PCR reagents must allow the reactions to proceed together in one tube. This prevents the use of optimal reagents and conditions for each individual reaction, potentially compromising reaction conditions and negatively impacting efficiency and yield.

[0005] Therefore, there is a need for improved methods for rapid detection of target RNA in a sample that allow for high efficiency and yield.

[0006] Summary of the Invention Provided herein is a method for detecting a target RNA in a sample. In some embodiments, provided herein is a method for detecting a target RNA in a sample, comprising: (a) providing a reaction mixture containing the sample, an amplification reagent, and a polymerase enzyme having both RNA- and DNA-dependent polymerase activity; (b) reverse transcribing the RNA to DNA by incubating for a reverse transcription time of 5 minutes or less; and (c) amplifying the DNA by performing a thermal cycling protocol that includes multiple amplification cycles, where each amplification cycle includes at least a thermal denaturation step and an annealing step.

[0007] In some embodiments, the amplification reagent comprises deoxynucleotide triphosphates, a buffer, cofactors, and an oligonucleotide primer configured to amplify a target RNA in a sample. In some embodiments, the oligonucleotide primer comprises a forward primer and a reverse primer. In some embodiments, the oligonucleotide primer is provided at a concentration of at least 6 μM (e.g., 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, or a range therebetween).

[0008] In some embodiments, the oligonucleotide primers are provided at a concentration of 12 μM. In some embodiments, the polymerase enzyme is provided at a concentration of at least 0.4 U / μL (e.g., 0.4 U / μL, 0.5 U / μL, 0.6 U / μL, 0.7 U / μL, 0.8 U / μL, 0.9 U / μL, 1.0 U / μL, 1.1 U / μL, 1.2 U / μL, or ranges therebetween). In some embodiments, the polymerase enzyme is provided at a concentration of 0.8 U / μL.

[0009] In some embodiments, the cofactor is a magnesium salt or a manganese salt. In some embodiments, the cofactor is a manganese salt. In some embodiments, the manganese salt is MnCl2. In some embodiments, the cofactor is provided at a concentration of 3 mM to 8 mM (e.g., 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, or ranges therebetween). In some embodiments, the cofactor is provided at a concentration of 4 mM.

[0010] In some embodiments, the reverse transcription time is 2 minutes or less. In some embodiments, the reverse transcription time is 30 seconds or less. In some embodiments, the reverse transcription time is 12 seconds or less. In some embodiments, the reverse transcription time is 5 seconds or less. In some embodiments, the reverse transcription time is 1 second or less.

[0011] In some embodiments, the reverse transcription step occurs at a temperature between 64 and 72°C (e.g., 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, or ranges therebetween). In some embodiments, the reverse transcription step occurs at a temperature of 68°C. In some embodiments, each thermal denaturation step occurs at 91-99°C (e.g., 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, or ranges therebetween) for 1 second. In some embodiments, each annealing step occurs at 64-72°C (e.g., 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, or ranges therebetween) for 4 seconds. In some embodiments, each thermal denaturation step occurs at 95°C for 1 second and each annealing step occurs at 68°C for 4 seconds. In some embodiments, the thermal cycling protocol comprises at least 30 (e.g., 30, 35, 40, 45, 50, 55, 60, or more, or ranges therebetween) amplification cycles. In some embodiments, the thermal cycling protocol comprises 40 amplification cycles.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Box plot of Cq when RT time of MS2 RT-qPCR is changed, the unit is seconds. The number of replicates is indicated on the graph.

[0013] [Figure 2] Mg 2+ Cofactor or Mn 2+ Amplification curves of MS2 RNA amplified with either cofactor.

[0014] [Figure 3] Mg 2+ Cofactor or Mn 2+ Amplification curves of the plasmid containing the closed HCV cDNA and RNA in vitro transcribed from the plasmid with either cofactor.

[0015] [Mode for carrying out the invention] definition Although any methods and materials similar or equivalent to those described herein can be used to practice or test the embodiments described herein, some suitable methods, compositions, devices, and materials are described herein. However, before describing the materials and methods of the present invention, it is to be understood that the present invention is not limited to the specific molecules, compositions, methods, or protocols described herein, which can be modified according to routine experimentation and optimization. It is also to be understood that the terminology used in the description is only for the purpose of describing the particular forms or embodiments, and is not intended to limit the scope of the embodiments described herein.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, shall control. Thus, in the context of the embodiments described herein, the following definitions apply.

[0017] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise, i.e., for example, reference to "a peptide molecule" is a reference to one or more peptide amphiphiles and equivalents thereof known to those skilled in the art.

[0018] As used herein, the term "comprise" and variations thereof indicate the presence of the recited feature(s), element(s), method step(s), etc., but do not exclude the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term "consisting of" and variations thereof indicate the presence of the recited feature(s), element(s), method step(s), etc., and excludes the presence of any unrecited feature(s), element(s), method step(s), etc., except for impurities normally associated therewith. The phrase "consisting essentially of" indicates the presence of the recited feature(s), element(s), method step(s), etc., and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using the open-ended term "comprise." Such embodiments encompass a number of limited "consisting of" and / or "consisting essentially of" embodiments, which limited embodiments may alternatively be claimed or described using that language.

[0019] As used herein, the term "analyzing" and equivalent language refers to any step taken to characterize a sample or one or more components thereof. Examples of analytical steps include, for example, quantification of sample components (e.g., target nucleic acids), sequencing of sample components, etc.

[0020] As used herein, the term "preparing" and equivalent language refers to any step taken to modify a sample or one or more of its components, e.g., for use in a subsequent analytical or detection step. Examples of sample preparation steps include, e.g., diluting or concentrating the sample, isolating or purifying a sample component, heating or cooling the sample, amplifying a sample component (e.g., a nucleic acid), labeling a sample component, and the like.

[0021] As used herein, the terms "sample" and "specimen" are used synonymously and in their broadest sense. In one sense, a sample is meant to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum, feces, urine, and the like. Environmental samples include environmental materials, such as surface materials, soil, mud, sludge, biofilms, water, and industrial samples. However, these examples should not be construed as limiting the types of samples to which the present invention can be applied.

[0022] The term "system" as used herein refers to a collection of compositions, devices, articles, materials, etc., grouped together in any suitable manner (e.g., physically associated; in fluid, electronic, or data communication; packaged together, etc.) for a particular purpose.

[0023] Detailed Description Provided herein is a method for detecting target RNA in a sample. The method described herein allows for rapid transcription and polymerase chain reaction using a single enzyme, instead of one enzyme for reverse transcription and one enzyme for polymerase chain reaction. The method includes: preparing a reaction mixture containing a sample, an amplification reagent, and a polymerase enzyme having both RNA- and DNA-dependent polymerase activity; reverse transcribing RNA to DNA by incubating for a reverse transcription time (e.g., 5 minutes or less); and amplifying DNA by performing a thermal cycling protocol that includes multiple amplification cycles, where each amplification cycle includes at least a thermal denaturation step and an annealing step.

[0024] A reaction mixture Any suitable PCR reagent can be used in the reaction mixture. Suitable PCR reagents include water, buffer, dNTP, primer, control, catalyst, initiator, promoter, cofactor, salt, chelator, probe, fluorescent dye, and combinations thereof. For example, the reaction mixture can contain amplification reagent. Amplification reagent can include dNTP, buffer, cofactor, and oligonucleotide primer configured to amplify target RNA in sample.

[0025] The terms "primer" and "oligonucleotide primer" are used interchangeably herein. In general, a primer is a shorter nucleic acid that is complementary to a longer template. During replication, a primer can be extended based on the template sequence to generate a longer nucleic acid that is a complementary copy of the template. Extension can occur by sequential addition of individual nucleotides (e.g., by the action of a polymerase). A primer can be DNA, RNA, an analog thereof (e.g., an artificial nucleic acid), or any combination thereof. A primer can have any suitable length. For example, a primer can be at least 10 nucleotides. For example, a primer can be at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides. Exemplary primers are chemically synthesized.

[0026] The oligonucleotide primers can be provided as at least one pair of primers for amplifying at least one nucleic acid target. For example, a pair of primers can be a forward primer (i.e., a sense primer) and a reverse primer (i.e., an antisense primer) that collectively define the opposite ends (and thus the length) of the resulting amplicon. Any suitable primer concentration can be used. In some embodiments, the oligonucleotide primers are provided at a concentration of at least 6 μM. For example, the oligonucleotide primers can be provided at a concentration of at least 6 μM, at least 7 μM, at least 8 μM, at least 9 μM, at least 10 μM, at least 11 μM, or at least 12 μM. In some embodiments, the oligonucleotide primers are provided at a concentration of 12 μM.

[0027] The polymerase enzyme may be any suitable enzyme having both RNA- and DNA-dependent polymerase activity. The polymerase enzyme having both RNA- and DNA-dependent polymerase activity may be a commercially available polymerase (e.g., from Boehringer Mannheim Corp., Indianapolis, Ind.; Life Technologies, Inc., Rockville, Md.; New England Biolabs, Inc., Beverley, Mass.; Perkin Elmer Corp., Norwalk, Conn.; Pharmacia LKB Biotechnology, Inc., Piscataway, NJ; Qiagen, Inc., Valencia, Calif.; Stratagene, La Jolla, Calif.). For example, the polymerase enzyme may be HawkZ05 Fast polymerase. Any suitable polymerase enzyme concentration may be used. In some embodiments, the polymerase enzyme may be provided at a concentration of at least 0.4 U / μL. For example, the polymerase enzyme may be provided at a concentration of at least 0.4 U / μL, at least 0.5 U / μL, at least 0.6 U / μL, at least 0.7 U / μL, or at least 0.8 U / μL. In some embodiments, the polymerase enzyme is provided at a concentration of 0.8 U / μL.

[0028] In some embodiments, the polymerase has both DNA- and RNA-dependent polymerase activity. In some embodiments, the polymerase is compatible with hot-start PCR. In some embodiments, the polymerase is part of an aptamer / enzyme system that allows for hot-start PCR (e.g., the polymerase is inactivated below a threshold temperature). In some embodiments, a polymerase from Thermus sp. Z05 is provided.

[0029] The cofactor may be any cofactor suitable for the polymerase enzyme used. For example, the cofactor may be a magnesium salt. For example, the magnesium salt may be MgCl2 or MgSO4. As another example, the cofactor may be a manganese salt. For example, the manganese salt may be MnCl2 or MnSO4. Any suitable cofactor concentration may be used. In some embodiments, the cofactor is provided at a concentration of 3 mM to 8 mM. For example, the cofactor may be provided at a concentration of 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, or 8 mM. In some embodiments, the cofactor is provided at a concentration of 4 mM.

[0030] According to the embodiments provided herein, the PCR reagents can also include one or more probes, or any nucleic acid to which at least one label, e.g., at least one dye, is attached. The probe can be a sequence-specific binding partner of the nucleic acid target and / or amplicon. The probe can be designed to enable detection of target amplification based on fluorescence resonance energy transfer (FRET) and includes one or more nucleic acids to which a pair of dyes is attached that collectively exhibit fluorescence resonance energy transfer (FRET) when in close proximity to each other. The pair of dyes can provide, among others, a first emitter and a second emitter, or an emitter and a quencher. The fluorescence emission from the pair of dyes changes when the dyes are separated from each other, such as by probe cleavage during primer extension (e.g., 5' nuclease assays using TAQMAN probes), or when the probe hybridizes to an amplicon (e.g., molecular beacon probes). The nucleic acid portion of the probe can have any suitable structure or origin, for example, the portion can be a locked nucleic acid, a member of a universal probe library, and the like. In other cases, the probe and one of the primers of a primer pair may be combined in the same molecule. For example, a primer-probe molecule may have a primer sequence at its 3' end and a molecular beacon-type probe at its 5' end. Using this arrangement, related primer-probe molecules labeled with different dyes can be used in a multiplex assay with the same reverse primer to quantify target sequences that differ by one nucleotide (single nucleotide polymorphisms (SNPs)).

[0031] B. Reverse transcription Reverse transcription refers to the process of generating a complementary DNA strand (cDNA) from an RNA template present in a sample. The methods described herein require a short incubation time to generate a cDNA product from an RNA template. In particular, the disclosed methods include reverse transcribing RNA to DNA by incubating for a reverse transcription time (e.g., 5 minutes or less). For example, the reverse transcription time may be 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, 90 seconds or less, 60 seconds or less, 30 seconds or less, 15 seconds or less, 12 seconds or less, 10 seconds or less, 8 seconds or less, 5 seconds or less, or 1 second or less. In some embodiments, the reverse transcription time is 0 seconds.

[0032] The reverse transcription step can occur at any suitable temperature depending on the polymerase enzyme used. In some embodiments, the reverse transcription step is performed at a higher temperature than temperatures typically used for reverse transcription methods. For example, HawkZ05 Fast polymerase is sold with an aptamer whose enzymatic activity is blocked below 55°C. Thus, for methods using HawkZ05 Fast polymerase, the reverse transcription step is performed at a temperature above 55°C. In some embodiments, the reverse transcription step may be performed at a temperature between 60-70°C. For example, the reverse transcription step may occur at a temperature above 55°C, above 60°C, or above 65°C. In some embodiments, the reverse transcription step occurs at a temperature of 68°C. Other polymerases may require alternative temperatures for the reverse transcription step.

[0033] Certain RNA targets require prior heat denaturation of the RNA before it is added to the RT-PCR reaction. For example, heat denaturation of rotavirus or the RNA secondary structure found in hepatitis C virus requires melting temperatures considerably higher than the optimal temperature range of commonly used reverse transcriptases. This results in heat denaturation of the reverse transcription polymerase enzyme. For example, commonly used reverse transcriptases such as Maloney murine leukemia virus (MMLV) reverse transcriptase or avian myeloblastosis virus (AMV) reverse transcriptase have an optimal temperature range of 37-42°C. Thus, targets such as rotavirus or hepatitis C require a preliminary RNA heat denaturation step, a process that is not compatible with the one-step closed cartridge design. In contrast, the methods described herein allow the reverse transcription step to be performed at a higher temperature range, such as the range of 60-70°C. This allows reverse transcription and subsequent detection of the RNA target without the need for preliminary RNA heat denaturation.

[0034] c. DNA amplification PCR reactions generally also involve a process of amplification, i.e., replication, which occurs repeatedly over time to produce multiple copies of at least one segment of a template molecule. Amplification generally relies on alternating cycles of heating and cooling (i.e., thermocycling) to achieve successive rounds of replication.

[0035] The method disclosed herein includes amplifying DNA by performing a thermal cycling protocol that includes multiple amplification cycles. Amplification can be performed using any suitable reagent as described above. Each amplification cycle includes at least a thermal denaturation step and an annealing step. For example, each amplification cycle may oscillate between two or more temperature set points, such as a high melting (denaturation) temperature and a low annealing / extension temperature. In other embodiments, each amplification cycle may oscillate between three or more temperature set points, such as a high melting temperature, a low annealing temperature, and an intermediate extension temperature.

[0036] Any suitable temperature and duration can be used for each step of the amplification cycle. Generally, each heat denaturation step is performed at a temperature between 91 and 98°C. For example, each heat denaturation step may be performed at 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, or 98°C. In some embodiments, each heat denaturation step is performed at 95°C. In some embodiments, each heat denaturation step may be performed for less than 20 seconds. For example, each heat denaturation step may be performed for less than 20 seconds, less than 10 seconds, less than 5 seconds, less than 4 seconds, less than 3 seconds, less than 2 seconds, or less than 1 second. In some embodiments, each heat denaturation step is performed at 95°C for 1 second.

[0037] Suitable annealing temperatures depend on the primer pair and may generally be performed at 45-70°C. For example, each annealing step may be performed at a temperature of 45°C, 50°C, 55°C, 58°C, 60°C, 65°C, or 68°C. Each annealing step may be performed for less than 20 seconds. For example, each annealing step may be performed for less than 20 seconds, less than 10 seconds, or less than 5 seconds. In some embodiments, each annealing step is performed at 68°C for 4 seconds.

[0038] In some embodiments, the thermal cycling protocol may include an initial high temperature hold phase (e.g., 95° C.) prior to performing multiple amplification cycles. For example, the thermal cycling protocol may include an initial high temperature hold phase at 95° C. for 2 minutes or less. In some embodiments, the thermal cycling protocol may include an initial hold phase at 95° C. for 2 minutes, 90 seconds, 1 minute, 30 seconds, or 15 seconds.

[0039] Amplification can result in an exponential or linear increase in copy number as it proceeds. Typical amplification results in a greater than 1,000-fold increase in copy number and / or signal. Any suitable number of amplification cycles can be performed to generate the desired signal. For example, the thermal cycling protocol can include at least 30 amplification cycles. In some embodiments, the thermal cycling protocol includes 40 amplification cycles.

[0040] d.Device The disclosed methods can be performed using any suitable device. For example, a device suitable for performing the disclosed RT-qPCR method may include a sample container, a first temperature zone, a second temperature zone, and a transport mechanism. The transport mechanism physically moves the sample container between the first and second temperature zones. The sample container may be a well that can contain a liquid sample. Alternatively, the sample container may be a porous material that can adsorb a liquid sample. Each temperature zone may include a temperature regulator that maintains a fixed temperature within the temperature zone. In some embodiments, a suitable device further includes a detection zone, e.g., a detection zone that includes a fluorometer. In some embodiments, one or both of the temperature zones may be a detection zone. Examples of devices are disclosed in International Application No. PCT / US2018 / 034443, the entire contents of which are incorporated herein by reference.

[0041] e.Kit The PCR reagents described herein may be incorporated into a kit for rapid detection of target RNA in a sample. For example, the disclosed components may be incorporated into a kit for rapid clinical diagnosis, e.g., for detecting viral RNA in a sample. A suitable kit may contain any suitable primers and / or probes for detecting any desired RNA in a sample. For example, a kit may include components suitable for viral RNA detection, which requires high temperatures (e.g., 60-70°C) for heat denaturation of the RNA. Such a kit would allow for rapid reverse transcription and amplification of RNA targets, such as rotavirus or hepatitis C virus, without the need for a preceding RNA heat denaturation step. In some embodiments, the kit may include suitable PCR reagents in a single sealed cartridge for detecting target RNA in a sample.

[0042] [Example] Example 1 Fast RT-qPCR assay method HawkZ05 Fast polymerase is commercially available as a fast RT-qPCR assay. The manufacturer recommends that the RT step should be performed for 2 to 5 min (4). We tested how reaction conditions, including higher levels of primers and enzymes, affected the RT-qPCR assay. Surprisingly, very similar Cq values ​​were measured when the RT time was 5 min, 2 min, 30 s, 12 s, and 5 s. RNA extracted from MS2 bacteriophage was amplified using MS2 RT-qPCR primers and probes adapted from Beck, et al. (5). PCR data were analyzed using LinRegPCR (6, 7). RT-qPCR was performed on 15 μl samples using a prototype instrument. The RT step consisted of 68°C from 0 s to 5 min, a 15 s hold at 95°C, followed by 40 cycles of 95°C for 1 s and 68°C for 4 s.

[0043] Reaction Composition The RT-qPCR reaction composition contained the following: 10% glycerol (ACROS) 0.2% Tween 20 (Pierce) 150 mM trehalose (Life Sciences Advanced Technologies) 6mg / ml ultrapure BSA (Ambion) 65 mM Tris pH 8.0 (Thermo Scientific) 62.4mM Bicine / KOH pH 8.0 (USB) 65 mM potassium glutamate (Sigma) 0.4mM dNTP (Thermo Scientific) 4.0 mM MnCl2 (Sigma) 12 μM MS2 forward and reverse primer mix (IDT; see sequence below) 500 nM MS2 FAM-labeled hydrolysis probe (IDT; see sequence below) 0.8U / μL HawkZ05 Fast polymerase (Roche Custom Biotech) Approximately 5,000 copies of MS2 RNA isolated from MS2 bacteriophage (Zeptometrix 0810066) using the Dynal MyOne Silane Viral Isolation kit (Thermo Scientific). Forward primer: 5'-agg tcg gta cta aca tca agt-3' Reverse primer: 5'-gat atg ttg cac gtt gtc tgg a-3' Hydrolysis probe: 5'- / 56-FAM / cgt ctg tcg / zen / tat cca gct gca aac t / 3IABkFQ-3' result There was no difference in the Cq measured in the RT-qPCR assays with RT lengths ranging from 5 s to 5 min (300 s) (Figure 1). Significant RT activity can be observed even when held at 68 °C for only 0 s. However, the difference between the average 12 s RT and the 0 s RT was 1.4 Cq. This corresponds to approximately 2.5-3 times less cDNA yield.

[0044] Surprisingly, 0 sec RT combined with fast PCR cycling conditions also yielded product. The first choice cofactor for Z05 was Mg 2+ and RT-PCR was performed using Mn 2+ Therefore, to test whether the Cq observed at 0 s RT was due to contaminating phage DNA, MS2 RNA was incubated with 1000 μl of Mg 2+ Amplify with cofactors and Mn 2+ The results were compared with those of the cofactors. 2+ The PCR curve for the cofactor reaction is shown in Fig. 2+ It rose out of background approximately six cycles sooner than the cofactor reaction (Figure 2). This difference in amplification corresponds to approximately two orders of magnitude difference in the amount of cDNA generated during the RT step. The absence of amplification in the no template control reaction (NTC) demonstrates that the reagents were not contaminated with MS2 DNA.

[0045] Similar studies were performed using Hepatitis C in vitro transcribed RNA, which had been treated with DNAase I as part of the transcription protocol (MEGAshortscript kit, Applied Biosystems). 2+ is Mn 2+ The other two curves are positive controls to demonstrate that the cofactor is acceptable for the PCR assay. 2+ RNA amplified using 100% Mg as a cofactor 2+ The reaction amplifies visibly much earlier than the reaction using Mn as a cofactor, and the estimated Cq is 2+ The ratio of percent RNA yield using the above methods appears to be 8-10.

[0046] It should be understood that the above detailed description and accompanying examples are merely illustrative and should not be taken as limitations on the scope of the present disclosure, which is defined solely by the appended claims and their equivalents.

[0047] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the disclosure, including but not limited to those related to the chemical structure, substituents, derivatives, intermediates, synthesis, compositions, formulations, or methods of use of the disclosure.

[0048] All patents and publications referenced herein are hereby incorporated by reference in their entirety.

[0049] reference The following references, some of which are cited above, are incorporated herein by reference in their entirety: 1. Farrar JS, Wittwer CT. Extreme PCR: efficient and specific DNA amplification in 15-60 seconds. Clinical chemistry. 2015;61(1):145-53. 2. Wittwer CT, Houskeeper JA, Myers Bigel PA, inventors;University of Utah Research Foundation, assignee. Extreme Reverse Transcription PCR2017 11 May 2017. 3. Mijatovic-Rustempasic S, Tam KI, Kerin TK, Lewis JM, Gautam R, Quaye O, et al. Sensitive and specific quantitative detection of rotavirus A by one-step real-time reverse transcription-PCR assay without antecedent double-stranded-RNA denaturation. Journal of clinical microbiology. 2013;51(9):3047-54. 4. Nakerakanti S, Sammeta N. Guidelines for the optimization of assays using HawkZ05 Fast Polymerase. In: Biotech RC, editor. 2018. 5. Beck ET, Jurgens LA, Kehl SC, Bose ME, Patitucci T, LaGue E, et al. Development of a rapid automated influenza A, influenza B, and respiratory syncytial virus A / B multiplex real-time RT-PCR assay and its use during the 2009 H1N1 swine-origin influenza virus epidemic in Milwaukee, Wisconsin. The Journal of molecular diagnostics: JMD. 2010;12(1):74-81. 6. Ramakers C, Ruijter JM, Deprez RH, Moorman AF. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neuroscience letters. 2003;339(1):62-6. 7. Ruijter JM, Ramakers C, Hoogaars WM, Karlen Y, Bakker O, van den Hoff MJ, et al. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic acids research. 2009;37(6):e45. [Brief description of the drawings]

[0050] [Figure 1] Box plot of Cq for MS2 RT-qPCR with varying RT times (units are seconds). The number of replicates is indicated on the graph. [Diagram 2] Amplification curves of MS2 RNA amplified with either Mg2+ or Mn2+ cofactor. [Diagram 3] Amplification curves of plasmids containing closed HCV cDNA and RNA in vitro transcribed from plasmids using either Mg2+ or Mn2+ cofactor.

Claims

1. 1. A method for detecting a target RNA in a sample, comprising: a. (i) the sample; (ii) an amplification reagent comprising: a deoxynucleotide triphosphate; a buffer; a cofactor selected from a magnesium salt or a manganese salt, the cofactor being provided at a concentration of 3 mM to 8 mM; and an oligonucleotide primer configured to amplify the target RNA, the oligonucleotide primer being provided at a concentration of at least 6 μM; and (iii) providing a reaction mixture containing a polymerase enzyme having both RNA- and DNA-dependent polymerase activity provided at a concentration of at least 0.4 U / μL; b. reverse transcribing the RNA into DNA by incubating at a temperature of 64-72° C. for a reverse transcription time of 30 seconds or less; c. amplifying the DNA by a thermal cycling protocol comprising a plurality of amplification cycles, each of which includes at least a thermal denaturation step at a temperature of 91-98° C. for less than 5 seconds, and an annealing step at a temperature of 45-70° C. for less than 10 seconds; The method comprising:

2. The method of claim 1 , wherein the oligonucleotide primers comprise a forward primer and a reverse primer.

3. 3. The method of claim 1 or 2, wherein the oligonucleotide primer is provided at a concentration of 12 μM.

4. 4. The method of claim 1, wherein the polymerase enzyme is provided at a concentration of 0.8 U / μL.

5. The method of claim 1 , wherein the cofactor is a manganese salt.

6. The manganese salt is MnCl ２ The method according to claim 5, wherein

7. 10. The method of claim 1, wherein the cofactor is provided at a concentration of 4 mM.

8. 8. The method of claim 1, wherein the reverse transcription time is 12 seconds or less.

9. The method of claim 8 , wherein the reverse transcription time is 5 seconds or less.

10. The method of claim 9 , wherein the reverse transcription time is 1 second or less.

11. 11. The method of any one of claims 1 to 10, wherein the reverse transcription occurs at a temperature of 68°C.

12. 12. The method of any one of claims 1 to 11, wherein each heat denaturation step is carried out at 95°C for 1 second and each annealing step is carried out at 68°C for 4 seconds.

13. 13. The method of any one of claims 1 to 12, wherein the thermal cycling protocol comprises at least 30 amplification cycles.

14. 14. The method of claim 13, wherein the thermal cycling protocol comprises at least 40 amplification cycles.

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