WO2020150442A1 - Rapid reverse transcription quantitative polymerase chain reaction - Google Patents
Rapid reverse transcription quantitative polymerase chain reaction Download PDFInfo
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- WO2020150442A1 WO2020150442A1 PCT/US2020/013827 US2020013827W WO2020150442A1 WO 2020150442 A1 WO2020150442 A1 WO 2020150442A1 US 2020013827 W US2020013827 W US 2020013827W WO 2020150442 A1 WO2020150442 A1 WO 2020150442A1
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- reverse transcription
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Definitions
- RNA in a sample may be used for clinical diagnostics, such as for the detection of a viral infection in a subject.
- RNA DNA copy
- RT-qPCR Quantitative or real-time PCR
- RT-qPCR assays can be performed in either a one-step or two-step reaction.
- cDNA synthesis and qPCR are performed in a single reaction vessel in a common reaction buffer.
- two-step RT-qPCR cDNA is synthesized in one reaction, and an aliquot of the cDNA is then used for a subsequent qPCR experiment.
- One-step reactions allow for minimal sample handling and closed-tube reactions, reducing chances for pipetting errors and cross-contamination.
- the combined RT and PCR reagents must allow these reactions to proceed together in one tube. This prevents use of the most optimal reagents and conditions for each individual reaction, thus potentially compromising reaction conditions and negatively affecting efficiency and yield.
- methods for detecting a target RNA in a sample comprising: (a) providing a reaction mixture containing the sample, amplification reagents, 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 no longer than 5 minutes; and (c) amplifying the DNA by performing a thermal cycling protocol comprising a plurality of amplification cycles, wherein each amplification cycle comprises at least a denaturation step and an annealing step.
- the amplification reagents comprise deoxynucleotide triphosphates, a buffer, a cofactor, and oligonucleotide primers configured for amplification of the target RNA in the sample.
- the oligonucleotide primers comprise a forward primer and a reverse primer.
- the oligonucleotide primers are provided at a concentration of at least 6mM (e.g., 6mM, 7mM, 8mM, 9mM, IOmM, 1 ImM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, or ranges therebetween).
- the oligonucleotide primers are provided at a concentration of 12mM.
- the polymerase enzyme is provided at a concentration of at least 0.4 U/pL (e.g., 0.4 U/pL, 0.5 U/pL, 0.6 U/pL, 0.7 U/pL, 0.8 U/pL, 0.9 U/pL, 1.0 U/pL, 1.1 U/pL, 1.2 U/pL, or ranges therebetween).
- the polymerase enzyme is provided at a concentration of 0.8 U/pL.
- 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 MnCh. In some embodiments, the cofactor is provided at a concentration of 3mM to 8mM (e.g., 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, or ranges therebetween). In some embodiments, the cofactor is provided at a concentration of 4mM.
- the reverse transcription time is no longer than 2 minutes. In some embodiments, the reverse transcription time is no longer than 30 seconds. In some embodiments, the reverse transcription time is no longer than 12 seconds. In some embodiments, the reverse transcription time is no longer than 5 seconds. In some
- the reverse transcribing step occurs at a temperature of 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
- each denaturation step is performed for 1 second 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).
- each annealing step is performed for 4 seconds 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).
- each denaturation step is performed for 1 second at 95 ° C and each annealing step is performed for 4 seconds at 68 ° C.
- the thermal cycling protocol comprises at least 30 amplification cycles (e.g., 30, 35, 40, 45, 50, 55, 60, or more, or ranges therebetween). In some embodiments, the thermal cycling protocol comprises 40 amplification cycles.
- Figure 1 Box and whisker plot of Cq of MS2 RT-qPCR with varying RT times in seconds. The number of replicates is indicated on graph.
- Figure 3 Amplification curves of a plasmid containing the closed HCV cDNA and in vitro transcribed RNA from the plasmid with either Mg 2+ or Mn 2+ cofactors.
- the term“comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc.
- the term “consisting of’ and linguistic variations thereof denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities.
- the phrase“consisting essentially of’ denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc.
- compositions, system, or method that do not materially affect the basic nature of the composition, system, or method.
- Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed“consisting of’ and/or“consisting essentially of’ embodiments, which may alternatively be claimed or described using such language.
- analyzing and linguistic equivalents thereof refers to any steps taken to a characterize a sample or one or more components thereof.
- exemplary analysis steps include, for example, quantification of a sample component (e.g., a target nucleic acid), sequencing a sample component, etc.
- sample preparation steps include, for example, dilution or concentration of a sample, isolation or purification of a sample component, heating or cooling a sample, amplification of a sample component (e.g., nucleic acid), labeling sample components, etc.
- a sample component e.g., nucleic acid
- sample and“specimen” are used interchangeably, and in the broadest senses.
- sample is meant to include a specimen or culture 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, stool, urine, and the like.
- Environmental samples include environmental material such as surface matter, soil, mud, sludge, biofilms, water, and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present invention.
- system 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.
- the methods described herein enable rapid transcription and polymerase chain reaction with a single enzyme, rather than one enzyme for reverse transcription and one enzyme for polymerase chain reaction.
- the methods comprise providing a reaction mixture containing the sample, amplification reagents, 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 (e.g., for no longer than 5 minutes); and amplifying the DNA by performing a thermal cycling protocol comprising a plurality of amplification cycles, wherein each amplification cycle comprises at least a denaturation step and an annealing step.
- PCR reagents include water, buffer, dNTPs, primers, controls, catalysts, initiators, promoters, cofactors, salts, chelating agents, probes, fluorescent dyes, and combinations thereof.
- the reaction mixture may contain amplification reagents.
- the amplification reagents may include dNTPs, a buffer, a cofactor, and oligonucleotide primers configured for amplification of the target RNA in the sample.
- a primer is a shorter nucleic acid that is complementary to a longer template.
- the primer may be extended, based on the template sequence, to produce a longer nucleic acid that is a complementary copy of the template. Extension may occur by successive addition of individual nucleotides (e.g., by the action of a polymerase).
- a primer may be DNA, RNA, an analog thereof (e.g., an artificial nucleic acid), or any combination thereof.
- a primer may have any suitable length.
- a primer may be at least 10 nucleotides.
- a primer may be at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides. Exemplary primers are synthesized chemically.
- Oligonucleotide primers may be supplied as at least one pair of primers for amplification of at least one nucleic acid target.
- a pair of primers may be a forward primer (i.e. a sense primer) and a reverse primer (i.e. an antisense primer) that collectively define the opposing ends (and thus the length) of a resulting amplicon.
- Any suitable concentration of primers may be used.
- the oligonucleotide primers are provided at a concentration of at least 6 mM.
- the oligonucleotide primers may be provided at a concentration of at least 6 mM, at least 7 pM, at least 8 pM, at least 9 pM, at least 10 pM, at least 11 pM, or at least 12 pM. In some embodiments, the oligonucleotide primers are provided at a concentration of 12 pM.
- the polymerase enzyme may be any suitable enzyme having both RNA and DNA-dependent polymerase activity.
- Polymerase enzymes having both DNA and RNA dependent polymerase activity may be commercially available polymerases (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, N.J.; Qiagen, Inc., Valencia, Calif.; Stratagene, La Jolla, Calif.).
- the polymerase enzyme may be HawkZ05 Fast Polymerase. Any suitable concentration of polymerase enzyme may be used.
- the polymerase enzyme may be provided at a concentration of at least 0.4 U/pL.
- the polymerase enzyme may be provided at a concentration of at least 0.4 U/pL, at least 0.5 U/pL, at least 0.6 U/pL, at least 0.7 U/pL, or at least 0.8 U/pL.
- the polymerase enzyme is provided at a concentration of 0.8 U/pL.
- 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., polymerase is inactivated below a threshold temperature). In some embodiments, a polymerase from Thermus species Z05 is provided.
- the cofactor may be any suitable cofactor for the polymerase enzyme used.
- the cofactor may be a magnesium salt.
- the magnesium salt may be MgCh or MgSCri.
- the cofactor may be a manganese salt.
- the manganese salt may be MnCh or MnSCri. Any suitable concentration of cofactor may be used.
- the cofactor is provided at a concentration of 3mM to 8mM.
- 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.
- PCR reagents can also include one or more probes, or any nucleic acid connected to at least one label, such as at least one dye.
- a probe may be a sequence-specific binding partner for a nucleic acid target and/or amplicon.
- the probe may be designed to enable detection of target amplification based on fluorescence resonance energy transfer (FRET), including one or more nucleic acids connected to a pair of dyes that collectively exhibit fluorescence resonance energy transfer (FRET) when proximate one another.
- FRET fluorescence resonance energy transfer
- the pair of dyes may provide first and second emitters, or an emitter and a quencher, among others.
- Fluorescence emission from the pair of dyes changes when the dyes are separated from one another, such as by cleavage of the probe during primer extension (e.g., a 5' nuclease assay, such as with a TAQMAN probe), or when the probe hybridizes to an amplicon (e.g., a molecular beacon probe).
- the nucleic acid portion of the probe may have any suitable structure or origin, for example, the portion may be a locked nucleic acid, a member of a universal probe library, or the like. In other cases, a probe and one of the primers of a primer pair may be combined in the same molecule.
- the primer-probe molecule may include a primer sequence at its 3’ end and a molecular beacon-style probe at its 5’ end.
- a primer sequence at its 3’ end and a molecular beacon-style probe at its 5’ end.
- related primer-probe molecules labeled with different dyes can be used in a multiplexed assay with the same reverse primer to quantify target sequences differing by a single nucleotide (single nucleotide polymorphisms (SNPs)).
- SNPs single nucleotide polymorphisms
- Reverse transcription refers to the process of generating a complementary DNA strand (cDNA) from the RNA template present in the sample.
- the methods described herein require a short incubation time to generate a cDNA product from the RNA template.
- the disclosed methods comprise reverse transcribing the RNA to DNA by incubating for a reverse transcription time (e.g., for no longer than 5 minutes).
- the reverse transcription time may be no longer than 5 minutes, no longer than 4 minutes, no longer than 3 minutes, no longer than 2 minutes, no longer than 90 seconds, no longer than 60 seconds, no longer than 30 seconds, no longer than 15 seconds, no longer than 12 seconds, no longer than 10 seconds, no longer than 8 seconds, no longer than 5 seconds, or no longer than 1 second.
- the reverse transcription time 0 seconds.
- the reverse transcription step can occur at any suitable temperature dependent on the polymerase enzyme used.
- the reverse transcription step is performed at an elevated temperature compared to the temperature typically used for methods of reverse transcription.
- the HawkZ05 Fast Polymerase is sold with aptamer that prevents enzymatic activity below 55°C. Accordingly, for methods using the HawkZ05 Fast polymerase the reverse transcription step is performed at a temperature above 55 ° C.
- the reverse transcription step may be performed at a temperature of 60-70 ° C.
- the reverse transcription step may occur at a temperature above 55 ° C, above 60 ° C, or above 65 ° C.
- the reverse transcription step occurs at a temperature of 68 ° C.
- Other polymerases may require alternative temperatures for the reverse transcription step.
- RNA targets require antecedent denaturation of the RNA prior to adding the RNA to the RT-PCR reaction.
- denaturation of rotavirus or the RNA secondary structure seen in hepatitis C virus requires melting temperatures significantly above the optimal temperature range of commonly used reverse transcription enzymes. This leads to denaturation of the reverse transcription polymerase enzyme.
- commonly used reverse transcription enzymes such as Maloney murine leukemia virus (MMLV) reverse transcriptase or avian myeloblastoma virus (AMV) reverse transcriptase have an optimal temperature range of 37-42 ° C.
- targets such as rotavirus or hepatitis C require a preliminary RNA denaturation step, a process which is not compatible for a one step, closed cartridge design.
- the methods described herein enable the reverse transcription step to be performed at an elevated temperature range, such as in the range of 60-70 ° C. This allows for reverse transcription and subsequent detection of RNA targets without the need for antecedent denaturation of the RNA.
- PCR reactions also generally involve a process of amplification, or a reaction in which replication occurs repeatedly over time to form multiple copies of at least one segment of a template molecule.
- amplification relies on alternating cycles of heating and cooling (i.e., thermal cycling) to achieve successive rounds of replication.
- the methods disclosed herein comprise amplifying the DNA by performing a thermal cycling protocol comprising a plurality of amplification cycles.
- the amplification may be performed using any suitable reagents as described above.
- Each amplification cycle comprises at least a denaturation step and an annealing step.
- each amplification cycle may alternate between two or more temperature set points, such as a higher melting (denaturation) temperature and a lower annealing/ extension temperature.
- each amplification cycle may alternate among three or more temperature set points, such as a higher melting temperature, a lower annealing temperature, and an intermediate extension temperature.
- each denaturation step is performed at a temperature from 91-98 ° C.
- each denaturation step may be performed at 91 °C, 92 °C, 93 °C, 94 °C, 95 °C, 96 °C, 97 °C, or 98 °C.
- each denaturation step is performed at 95 °C.
- each denaturation step may be performed for less than 20 seconds.
- each 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 1 second.
- each denaturation step is performed for 1 second at 95 ° C.
- the appropriate annealing temperature is dependent on the primer pair and may generally be performed at 45-70 ° C.
- 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.
- each annealing step may be performed for less than 20 seconds, less than 10 seconds, or less than 5 seconds.
- each annealing step is performed for 4 seconds at 68 ° C.
- the thermal cycling protocol may comprise an initial hold at a high temperature (e.g. 95 ° C) prior to performing the plurality of amplification cycles.
- a high temperature e.g. 95 ° C
- the thermal cycling protocol may comprise an initial hold at 95 ° C for 2 minutes or less.
- the thermal cycling protocol may comprise an initial hold for 2 minutes, 90 seconds, 1 minute, 30 seconds, or 15 seconds at 95 ° C.
- Amplification may generate an exponential or linear increase in the number of copies as amplification proceeds. Typical amplifications produce a greater than 1,000-fold increase in copy number and/or signal. Any suitable number of amplification cycles may be performed to generate the desired signal.
- the thermal cycling protocol may comprise at least 30 amplification cycles. In some embodiments, the thermal cycling protocol comprises 40 amplification cycles. d. Devices
- a suitable device for performing the disclosed RT-qPCR methods may comprise a sample container, a first temperature zone, a second temperature zone, and a shuttling mechanism.
- the shuttling mechanism physically moves the sample container between the first and second temperature zones.
- the sample container may be a well capable of containing a liquid sample.
- the sample container may be a porous material capable of adsorbing a liquid sample.
- Each temperature zone may contain a temperature regulator that maintains a fixed temperature within a temperature zone.
- suitable devices further comprise a detection zone, such as a detection zone comprising a fluorometer.
- one or both of the temperature zones may be a detection zone. Exemplary devices are described in International Application No. PCT/US2018/034443, the entire contents of which are incorporated herein by reference. e. Kits
- PCR reagents described herein may be incorporated into a kit for rapid detection of a target RNA in a sample.
- the disclosed components may be incorporated into a kit for rapid clinical diagnostics, such as for detection of viral RNA in sample.
- kits may contain any appropriate primers and/or probes for detection of any desired RNA in the sample.
- the kit may contain the appropriate components for detection of viral RNA that requires elevated temperatures (e.g. 60-70 ° C) for denaturation of the RNA.
- elevated temperatures e.g. 60-70 ° C
- kits would enable rapid reverse transcription and amplification of RNA targets such as rotavirus or hepatitis C virus without the need for an antecedent RNA denaturation step.
- the kit may comprise the appropriate PCR reagents in a single closed cartridge for the detection of target RNA in a sample.
- HawkZ05 Fast Polymerase is marketed as a fast RT-qPCR assay.
- the manufacturer recommends performing the RT step for 2 to 5 minutes (4). It was tested how reaction conditions involving higher levels of primer and enzyme would affect the RT-qPCR assay. Surprisingly, very similar Cqs were measured when the RT time was 5 min, 2 min, 30 sec.,
- MS2 RT-qPCR primers and probes adapted from Beck, et al. (5) were used to amplify RNA extracted from MS2 bacteriophage.
- LinRegPCR 6, 7
- a prototype instrument was used to perform RT-qPCR with 15 pi samples. The RT step was performed at 68 ° C from 0 seconds to 5 minutes, a 15 second hold at 95 ° C and then 40 cycles of 1 second at 95 ° C and 4 seconds at 68 ° C.
- the RT-qPCR reaction composition included the following:
- MS2 RNA isolated from MS2 bacteriophage Zeptometrix 0810066
- Dynal MyOne Silane Viral Isolation kit Thermo Scientific
- Reverse primer 5’ - gat atg ttg cac gtt gtc tgg a-3’
- Z05’s cofactor of choice is Mg 2+ for PCR and Mn 2+ for RT-PCR. Therefore, to test if contaminating phage DNA were responsible for the Cq observed with 0 seconds RT, the MS2 RNA was amplified using the Mg 2+ cofactor and compared the results to the Mn 2+ cofactor.
- the PCR curve of the Mn 2+ cofactor reaction rose up out of the background ⁇ 6 cycles before the Mg 2+ cofactor reaction Figure 2). This difference in amplification corresponds to ⁇ 2 orders of magnitude difference in amount of cDNA produced during RT step.
- NTC No Template Control reaction
- Amplification efficiency linking baseline and bias in the analysis of quantitative PCR data. Nucleic acids research. 2009;37(6):e45.
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Priority Applications (7)
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US17/421,677 US20220064707A1 (en) | 2019-01-17 | 2020-01-16 | Rapid reverse transcription quantitative polymerase chain reaction |
EP20741854.2A EP3911755A4 (en) | 2019-01-17 | 2020-01-16 | Rapid reverse transcription quantitative polymerase chain reaction |
MX2021008567A MX2021008567A (en) | 2019-01-17 | 2020-01-16 | Rapid reverse transcription quantitative polymerase chain reaction. |
AU2020208417A AU2020208417A1 (en) | 2019-01-17 | 2020-01-16 | Rapid reverse transcription quantitative polymerase chain reaction |
CA3126398A CA3126398A1 (en) | 2019-01-17 | 2020-01-16 | Rapid reverse transcription quantitative polymerase chain reaction |
JP2021541470A JP2022517278A (en) | 2019-01-17 | 2020-01-16 | Rapid reverse transcription Quantitative polymerase chain reaction |
CN202080015004.0A CN113795593A (en) | 2019-01-17 | 2020-01-16 | Fast reverse transcription quantitative polymerase chain reaction |
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US201962793701P | 2019-01-17 | 2019-01-17 | |
US62/793,701 | 2019-01-17 |
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US20140005066A1 (en) * | 2012-06-29 | 2014-01-02 | Advanced Liquid Logic Inc. | Multiplexed PCR and Fluorescence Detection on a Droplet Actuator |
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WO2018136404A1 (en) * | 2017-01-19 | 2018-07-26 | Asuragen, Inc. | Methods of rna amplification |
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JP5191041B2 (en) * | 2007-04-05 | 2013-04-24 | エフ.ホフマン−ラ ロシュ アーゲー | Rapid one-step RT-PCR |
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ES2533732T3 (en) * | 2010-10-04 | 2015-04-14 | F. Hoffmann-La Roche Ag | Method for cell lysis in an RT-PCR reaction buffer |
WO2013177429A2 (en) * | 2012-05-24 | 2013-11-28 | University Of Utah Research Foundation | Extreme pcr |
CN107787370A (en) * | 2015-05-28 | 2018-03-09 | 卢西根公司 | RNA Molecular Detection |
US10900074B2 (en) * | 2015-11-05 | 2021-01-26 | University Of Utah Research Foundation | Extreme reverse transcription PCR |
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2020
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US20150140548A1 (en) * | 2012-05-21 | 2015-05-21 | Vela Operations Pte. Ltd. | Extraction control for rna |
US20140005066A1 (en) * | 2012-06-29 | 2014-01-02 | Advanced Liquid Logic Inc. | Multiplexed PCR and Fluorescence Detection on a Droplet Actuator |
WO2018136404A1 (en) * | 2017-01-19 | 2018-07-26 | Asuragen, Inc. | Methods of rna amplification |
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EP3911755A1 (en) | 2021-11-24 |
MX2021008567A (en) | 2021-09-21 |
EP3911755A4 (en) | 2022-10-19 |
CN113795593A (en) | 2021-12-14 |
CA3126398A1 (en) | 2020-07-23 |
JP2022517278A (en) | 2022-03-07 |
AU2020208417A1 (en) | 2021-08-05 |
US20220064707A1 (en) | 2022-03-03 |
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