WO2021192320A1 - 新型コロナウイルスの検査方法および検査試薬 - Google Patents

新型コロナウイルスの検査方法および検査試薬 Download PDF

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WO2021192320A1
WO2021192320A1 PCT/JP2020/014415 JP2020014415W WO2021192320A1 WO 2021192320 A1 WO2021192320 A1 WO 2021192320A1 JP 2020014415 W JP2020014415 W JP 2020014415W WO 2021192320 A1 WO2021192320 A1 WO 2021192320A1
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sample
pcr
mixed solution
solution
probe
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French (fr)
Japanese (ja)
Inventor
慎一郎 小林
四方　正光
健二 二宮
直子 高岡
英明 丸瀬
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Shimadzu Corp
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Shimadzu Corp
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Priority to PCT/JP2020/014415 priority Critical patent/WO2021192320A1/ja
Priority to PCT/JP2020/037492 priority patent/WO2021192370A1/ja
Priority to US17/912,379 priority patent/US20250059613A1/en
Priority to JP2022510682A priority patent/JPWO2021193853A1/ja
Priority to TW110110913A priority patent/TWI874625B/zh
Priority to PCT/JP2021/012663 priority patent/WO2021193853A1/ja
Publication of WO2021192320A1 publication Critical patent/WO2021192320A1/ja
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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
    • 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
    • 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]
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a method for testing a new type of coronavirus and a kit for carrying out the method.
  • Coronavirus is known to infect all animals including livestock, laboratory animals, pets and wild animals and cause various diseases. Among them, four types of coronavirus that infect humans and two types of severe pneumonia virus that infect from animals are known so far. The coronavirus that causes the common cold is said to account for 10 to 15% of colds and 35% during the epidemic. HCoV-229E and HCoV-OC43 were discovered in these coronaviruses in the 1960s, and two types, HCoV-NL63 and HCoV-HKU1, were discovered in the 2000s.
  • SARS coronavirus discovered in 2002 (hereinafter sometimes referred to as SARS-CoV) is considered to be a natural host of the Greater Horseshoe Bat. SARS-CoV is known to cause severe acute respiratory syndrome and cause serious symptoms in the human body.
  • MERS coronavirus discovered in 2012 (hereinafter sometimes referred to as MERS-CoV) originates from dromedary syndrome and is known to cause severe pneumonia when infected with humans. Both coronaviruses caused a worldwide epidemic and caused many infections.
  • SARS-CoV-2 coronavirus
  • SARS-CoV-2 a new type of coronavirus
  • the first epidemic is said to be in Wuhan City, Hubei province, China, but after that, it became a global epidemic, and the infection continued to spread to Southeast Asia, the Middle East, Europe, and the United States, mainly in East Asia.
  • the outbreak in Brazil in early 2020 spread the infection to all five continents except Antarctica.
  • the new coronavirus has not yet been established for effective prevention and treatment, and in some people it can cause severe pneumonia and, in the worst case, death, but the symptoms are not specific. No. For example, it presents with a wide range of symptoms, from asymptomatic to severe pneumonia and death. Typical symptoms are said to include fever, dry cough, fatigue, sputum, shortness of breath, sore throat, headache, myalgia or arthralgia. The initial symptoms are similar to the common cold and are difficult to distinguish in the early stages of onset, and slight fever and cold symptoms may persist for about a week after the latent period of infection, but the patient's initial symptoms. Symptoms are not limited to fever and cough peculiar to pneumonia, but may also be gastrointestinal and nervous system symptoms such as diarrhea, nausea, headache and general fatigue, making early diagnosis difficult. ing.
  • PCR polymerase chain reaction
  • Non-Patent Document 1 RNA is extracted from a sample, and the extracted RNA is amplified by a reverse transcription polymerase chain reaction (hereinafter, may be referred to as RT-PCR method) to detect SARS-CoV-2.
  • RT-PCR method reverse transcription polymerase chain reaction
  • SARS-CoV-2 In the case of SARS-CoV-2, it has been reported that the incubation period before the onset is more than one week and that it causes secondary infection during the incubation period, so it is a quick and accurate test. A method is desired. Further, in view of the worldwide epidemic of SARS-CoV-2, an inspection kit capable of relatively easily and highly accurate inspection of SARS-CoV-2 is also desired.
  • An object of the present invention is to provide a method for quickly and inexpensively inspecting the presence or absence of SARS-CoV-2 infection and a kit capable of easily carrying out the method.
  • the present invention A step of obtaining a mixed solution by mixing 3 ⁇ L to 5 ⁇ L of a sample sample collected from a subject or a mixed solution of the sample sample and a medium and 3 ⁇ L to 5 ⁇ L of a sample treatment solution containing sodium hydroxide as a main component.
  • the step of incubating the above mixed solution A step of adding a master mix containing a reaction solution, a primer, a probe, a reverse transcriptase, and a PCR enzyme to the mixed solution after the incubation to obtain a final mixed solution.
  • the inspection method of the new coronavirus having.
  • the present invention relates to a kit for testing a new type of coronavirus having a sample treatment solution containing sodium hydroxide as a main component, a reaction solution, a primer, a probe, a reverse transcriptase, and a PCR enzyme.
  • the RT-PCR method includes a 1-step RT-PCR method and a 2-step RT-PCR method, but the 1-step RT-PCR method is convenient from the viewpoint of suppressing contamination between samples, and is preferable.
  • the method for testing a new type of coronavirus of the present invention includes a step of mixing a sample sample collected from a subject or a mixed solution of the sample sample and a medium with a sample processing solution containing sodium hydroxide as a main component.
  • Specimen samples collected from the subject include pharyngeal swab, nasal swab, sputum, bronchial lavage fluid and the like.
  • the medium contains a virus storage solution and the like.
  • the medium used provides a growth environment for the culture target in culturing microorganisms and biological tissues, and a transport / storage medium such as a commercially available UTM medium (manufactured by Nippon Becton Dickinson Co., Ltd.) can be preferably used.
  • the sample sample may be mixed with phosphate buffered saline (hereinafter, may be referred to as PBS) or the like in addition to the medium.
  • PBS phosphate buffered saline
  • the sample treatment solution is an aqueous solution containing sodium hydroxide as a main component, and is added for the purpose of extracting RNA from a virus.
  • the sample treatment solution may be a metal chelating agent such as glycol ether dithiothreitol (hereinafter, may be referred to as EGTA) from the viewpoint of efficiently performing RT-PCR treatment described later to improve test accuracy. It may contain a reducing agent such as dithiothreitol (hereinafter, may be referred to as DTT).
  • the volume of the sample solution is 1, and the volume ratio of the sample processing solution is 0. Mix 4 to 2.3 times. It is considered that the genomic RNA of the virus can be extracted by obtaining the mixed solution mixed in the above volume ratio, and the reverse transcription reaction and the PCR reaction can be appropriately performed.
  • the mixing ratio of the sample liquid and the sample treatment liquid is preferably 0.8 to 1.3 times, more preferably 0.9 to 1.1 times, the volume ratio of the sample treatment liquid, where the volume of the sample liquid is 1.
  • the sample solution and the sample processing solution are mixed at the above mixing ratio, but the volume of the final mixed solution described later is preferably about 25 ⁇ L or less from the viewpoint that it can be easily handled with a small amount of sample sample.
  • the volume of the final mixed solution is 25 ⁇ L or less
  • the sample solution is mixed with 3 ⁇ L to 5 ⁇ L and the sample treatment solution is mixed with 3 ⁇ L to 5 ⁇ L to obtain a mixed solution. 5 ⁇ L is more preferable for both the sample solution and the sample processing solution.
  • the resulting mixture is incubated.
  • the incubation temperature is set as appropriate. From the standpoint of speed of inspection and accuracy of the results obtained, the temperature is preferably room temperature to 95 ° C., preferably 80 to 95 ° C., and the time is preferably 3 to 5 minutes.
  • the room temperature is usually around 25 ° C.
  • a master mix containing a reaction solution, a primer, a probe, a reverse transcriptase, and a PCR enzyme is added to the mixed solution that has undergone the above incubation step to obtain a final mixed solution.
  • the reaction solution contains a PCR buffer solution containing a surfactant.
  • the surfactant can be selected from anionic surfactants, cationic surfactants, amphoteric surfactants or nonionic surfactants. It is preferably a nonionic surfactant and preferably has a concentration of 0.05 to 5% (w / v).
  • the PCR buffer solution contains a mix of KCl, MgCl 2 and deoxyribonucleoside 5'-triphosphate (hereinafter, may be abbreviated as dNTP) from the viewpoint of performing efficient RT-PCR.
  • the dNTP mix is defined as deoxyadenosine triphosphate (hereinafter, may be abbreviated as dATP), deoxyguanosine triphosphate (hereinafter, may be abbreviated as dGTP), deoxycytidine triphosphate (hereinafter, may be abbreviated as dGTP), and deoxycytidine triphosphate (hereinafter, may be abbreviated as dATP).
  • dTTP deoxycytidine triphosphate
  • the PCR buffer solution is preferably Tris-hydrochloric acid from the viewpoint of performing efficient RT-PCR.
  • concentrations of dNTP, MgCl 2 , KCl and the buffer solution can be appropriately set according to the RT-PCR treatment described later.
  • MgCl 2 has a concentration of about 1.5 mM
  • KCl has a concentration of about 35 mM
  • dNTP has a concentration of about 200 ⁇ M
  • Tris-hydrochloric acid has a concentration of about 10 mM.
  • the PCR buffer can be used for PCR such as biologically-derived negatively charged substances that adsorb to DNA polymerase such as certain sugars and dyes, and biologically-derived positively charged substances that adsorb to DNA such as certain proteins. It contains a substance that binds to a substance that inhibits and neutralizes the PCR inhibitory action of the negatively charged substance and the positively charged substance.
  • a gene amplification reagent Ampdirect registered trademark, Shimadzu Corporation
  • Ampdirect Plus registered trademark, Shimadzu Corporation
  • a primer specific to the RNA sequence of SARS-CoV-2 can be used.
  • the primers listed in the table below can be exemplified.
  • PCR products are monitored by real-time measurement in the RT-PCR reaction described below.
  • the steps of detecting RT-PCR and the RT-PCR product are performed in the same container.
  • Real-time measurement of PCR products is also called real-time PCR.
  • PCR amplification products are usually detected by fluorescence. Fluorescence detection methods include a method using an intercalator fluorescent dye and a method using a fluorescently labeled probe.
  • the intercalating fluorescent dye for example, SYBR® Green I is used.
  • the intercalator fluorescent dye binds to the double-stranded DNA synthesized by PCR and fluoresces when irradiated with excitation light. By measuring this fluorescence intensity, the amount of PCR amplification product produced can be measured.
  • a probe labeled with one or more fluorescent dyes is added in the new coronavirus test method of the present invention.
  • the probe include a hydrolysis probe, Molecular Beacon, and the like.
  • the hydrolysis probe is an oligonucleotide with a fluorescent dye at the 5'end and a quencher at the 3'end.
  • the hydrolyzed probe specifically hybridizes to the template DNA in the PCR annealing step, but the presence of a quencher on the probe suppresses the generation of fluorescence even when irradiated with excitation light.
  • the fluorescent dye is released from the probe and the quencher is used.
  • the suppression of fluorescence generation is released and fluorescence is emitted.
  • the amount of amplification product produced can be measured.
  • 6-carboxyfluorescein 6-carboxy-X-rhodamine
  • Cyanine dye 4,7,2', 4', 5', 7'-hexachloro- 6-carboxyfluorescein etc.
  • quencher examples include TAMRA®, Black Hole Quencher (BHQ, registered trademark) 1, BHQ2, MGB-Eclipse®, DABCYL and the like.
  • the reverse transcriptase is an enzyme that produces single-stranded complementary DNA (cDNA) using coronavirus RNA as a template.
  • RNA dependence derived from RNA viruses such as Avian Myeloblastosis Virus (AMV), Moloney Murine Leukemia Virus (M-MLV) and Human Immunodeficiency Virus (HIV) DNA polymerases as well as variants thereof can be used.
  • AMV Avian Myeloblastosis Virus
  • M-MLV Moloney Murine Leukemia Virus
  • HV Human Immunodeficiency Virus
  • the reverse transcriptase is preferably an enzyme having an activity of 200 U or more.
  • the DNA polymerase that is a PCR enzyme is preferably a thermostable DNA polymerase derived from a thermophilic bacterium, and examples thereof include Taq, Tth, KOD, Pfu, and variants thereof.
  • Hot-start DNA polymerase may be used in view of avoiding non-specific amplification by DNA polymerase.
  • examples of the hot-start DNA polymerase include a DNA polymerase to which an anti-DNA polymerase antibody is bound or a DNA polymerase in which an enzyme active site is heat-sensitively chemically modified, and a DNA polymerase to which an anti-DNA polymerase antibody is bound is preferable.
  • the PCR enzyme is preferably an enzyme having an activity of 3 U or more.
  • a master mix containing the reaction, primers, probes, reverse transcriptase, and PCR enzyme is added to the incubated mixture to give the final mixture.
  • the master mix is preferably added in the range of 14 to 16 ⁇ L.
  • the final mixture obtained is subjected to RT-PCR treatment.
  • the reaction temperature conditions for the reverse transcription reaction in RT-PCR and the PCR conditions are appropriately set.
  • the RT-PCR treatment is carried out in a commercially available reaction tube for RT-PCR.
  • the presence or absence of SARS-CoV-2 can be determined in real time by real-time PCR, and rapid testing for the new coronavirus can be performed. can.
  • the progress of PCR can be confirmed in real time by monitoring the amplification curve of the PCR product using a fluorescent filter corresponding to the fluorescent dye used. If the fluorescence intensity increases with the number of PCR cycles, the presence of SARS-CoV-2 in the sample sample is determined to be positive, while if the fluorescence intensity does not increase in PCR, it is determined to be negative. NS.
  • the internal control nucleic acid is added to the master mix, the possibility of false negatives can be easily eliminated if the fluorescence intensity of the internal control nucleic acid increases according to the number of PCR cycles. Examples of the internal control may be those that do not affect the amplification of SARS-CoV-2, and may have sequences derived from other organisms or artificially designed sequences.
  • the present invention further provides a kit for testing a new type of coronavirus having a sample treatment solution containing sodium hydroxide, a reaction solution, a primer, a probe, a reverse transcriptase, and a PCR enzyme. ..
  • the above-mentioned test kit makes it possible to efficiently perform the test when a very small amount of sample is collected and the new coronavirus is tested according to each of the above-mentioned steps.
  • the sample treatment solution, reaction solution, primer, probe, reverse transcriptase, and PCR enzyme are as described above.
  • the above test kit may contain the sample treatment solution, reaction solution, primer, probe, reverse transcriptase, and PCR enzyme in different containers, but each of them follows the procedure of the test method for the new coronavirus of the present invention. It is preferable to premix a predetermined amount of the virus in advance because it is possible to avoid the complexity of mixing at the time of inspection.
  • the sample treatment solution may be mixed in one container with the reaction solution, the primer, the probe, the reverse transcriptase, and the PCR enzyme in predetermined amounts to form one container, or the reaction solution, the primer, and the probe may be mixed in each container.
  • the reverse transcriptase and the PCR enzyme may be mixed in a predetermined amount to form one container, and the reverse transcriptase and the PCR enzyme may be mixed in a predetermined amount to form another container.
  • the presence or absence of SARS-CoV-2 infection can be quickly and easily tested. Since the RT-PCR test has higher detection sensitivity than immunochromatography, it can provide a highly sensitive coronavirus test in a short time even for those who are infected but do not have symptoms such as high fever or cough. ..
  • the above-mentioned mixed solution was mixed with a vortex mixer, incubated by heat treatment for 5 minutes in a constant temperature device at 90 ° C., and then ice-cooled.
  • Reagent A Reaction solution containing PCR buffer containing surfactant
  • Reagent B Primer and probe
  • Reagent C 250 U reverse transcriptase and 3.125 U PCR enzyme
  • the above master mix of 15 ⁇ L was added to a PCR reaction tube containing 10 ⁇ L of the sample after incubation. Then, the mixture was mixed with a vortex mixer, the PCR reaction tube was set in the real-time PCR device, and the PCR reaction was started immediately.
  • the setting conditions for real-time RT-PCR are as follows. RT-PCR setting conditions Hold at 50 ° C. for 10 minutes and then at 95 ° C. for 1 minute. Then, after holding at 95 ° C. for 5 seconds, the temperature was lowered to 60 ° C. and held for 30 seconds for 45 cycles.
  • the SARS-CoV-2 test may handle a sample containing a medium, but if the sample containing the medium is added directly to the reaction solution, it is considered to have an effect on PCR.
  • the test method for the new coronavirus of the present invention eliminates such an influence, and the result of the presence or absence of the new coronavirus can be obtained quickly.
  • Step of incubating the above mixture A step of adding 14 ⁇ L to 16 ⁇ L of a master mix containing a reaction solution, a primer, a probe, a reverse transcriptase, and a PCR enzyme to the mixed solution after the incubation to obtain a final mixed solution.
  • the RT-PCR test has a higher detection sensitivity than immunochromatography, so even if you are infected but do not have symptoms such as high fever or cough, you can provide a highly sensitive coronavirus test in a short time. can.
  • a kit for testing a new type of coronavirus having a sample treatment solution containing sodium hydroxide as a main component, a reaction solution, a primer, a probe, a reverse transcriptase, and a PCR enzyme.
  • the sample processing solution is stored in one container, the reaction solution is stored in one container, the primer and the probe are stored in one container, and the reverse transcriptase and the PCR enzyme are stored in one container [7].
  • a method for inspecting the presence or absence of SARS-CoV-2 infection can be rapidly performed.
  • the RT-PCR test has a higher detection sensitivity than immunochromatography, so even if you are infected but do not have symptoms such as high fever or cough, you can test for highly sensitive coronavirus in a short time. be able to.

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PCT/JP2020/014415 2020-03-27 2020-03-27 新型コロナウイルスの検査方法および検査試薬 Ceased WO2021192320A1 (ja)

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PCT/JP2020/014415 WO2021192320A1 (ja) 2020-03-27 2020-03-27 新型コロナウイルスの検査方法および検査試薬
PCT/JP2020/037492 WO2021192370A1 (ja) 2020-03-27 2020-10-02 新型コロナウイルスの検査方法および検査試薬
US17/912,379 US20250059613A1 (en) 2020-03-27 2021-03-25 Method and reagent for testing novel coronavirus
JP2022510682A JPWO2021193853A1 (https=) 2020-03-27 2021-03-25
TW110110913A TWI874625B (zh) 2020-03-27 2021-03-25 新型冠狀病毒的檢測方法和檢測試劑
PCT/JP2021/012663 WO2021193853A1 (ja) 2020-03-27 2021-03-25 新型コロナウイルスの検査方法および検査試薬

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WO2007052765A1 (ja) * 2005-11-02 2007-05-10 Shimadzu Corporation Rnaの抽出方法及びrnaの検出方法

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