WO2022024934A1 - Improved method for detecting virus - Google Patents

Improved method for detecting virus Download PDF

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WO2022024934A1
WO2022024934A1 PCT/JP2021/027383 JP2021027383W WO2022024934A1 WO 2022024934 A1 WO2022024934 A1 WO 2022024934A1 JP 2021027383 W JP2021027383 W JP 2021027383W WO 2022024934 A1 WO2022024934 A1 WO 2022024934A1
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virus
rna
acid
group
coronavirus
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PCT/JP2021/027383
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French (fr)
Japanese (ja)
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奈々 山越
旦 岡山
謙太 寺内
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東洋紡株式会社
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Priority to CN202180059316.6A priority Critical patent/CN116529362A/en
Priority to JP2022540253A priority patent/JPWO2022024934A1/ja
Publication of WO2022024934A1 publication Critical patent/WO2022024934A1/en

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • 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
    • 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 detecting RNA virus by nucleic acid amplification. More specifically, without isolating and purifying nucleic acid from the sample, a mixed solution containing the sample, an anionic polymer, and a polar organic solvent is prepared and then heat-treated, and a real-time reverse transcription polymerase chain reaction (qRT-PCR) is performed. ) Is related to the detection of RNA virus by adding the reaction solution.
  • qRT-PCR real-time reverse transcription polymerase chain reaction
  • INDUSTRIAL APPLICABILITY According to the present invention, for example, RNA virus contained in saliva, pharyngeal swab, nasal swab, biological sample such as sputum, fecal sample, blood sample, environmental wiping sample, etc. can be detected with high sensitivity. Is.
  • the present invention can be used for life science research, clinical diagnosis, food hygiene inspection, environmental inspection, and the like
  • Viruses are roughly classified into DNA viruses having deoxyribonucleic acid as genomic nucleic acids and RNA viruses having ribonucleic acids. Viruses are known to have a high mutation rate due to their short generation time, but RNA viruses are particularly susceptible to mutation. It is known that such virus mutations have a great influence on the infectivity to the host and the type and severity of symptoms at the time of infection. Therefore, it is important to develop a method for promptly and accurately detecting even a mutated virus in order to prevent the spread of infectious diseases and to contain them.
  • Coronavirus is a causative virus that causes respiratory infections including colds, and it is said that about 10 to 35% of coronaviruses are caused by the coronavirus during the cold season. It is also known that mutant viruses occur, and rarely SARS (severe acute respiratory syndrome) coronavirus, MERS (Middle East respiratory syndrome) coronavirus, and new coronavirus infection (COVID-19) coronavirus (SARS). It is known that those that cause fatal and serious respiratory diseases such as -nCOV-2) occur. Therefore, it goes without saying that simple, rapid, and highly sensitive detection of coronavirus is important in clinical diagnosis, food hygiene inspection, environmental inspection, and the like.
  • Non-Patent Document 3 Non-Patent Document 4
  • Non-Patent Document 5 a method for detecting SARS-nCOV-2 is described in the "Pathogen Detection Manual 2019-nCoV" of the National Institute of Infectious Diseases. In these techniques, detection of coronavirus contained in a sample involves extraction and purification of viral RNA from the sample.
  • Non-Patent Document 6 K. Kang et al. Report that highly pathogenic North American pig genital respiratory syndrome viral RNA can be detected directly from pig serum samples by RT-PCR (Non-Patent Document 6).
  • RT-PCR reaction inhibitors vary greatly depending on the type of sample. For example, a large amount of PCR reaction inhibitor such as polysaccharide and digestive enzyme RNase is introduced into saliva sample.
  • PCR reaction inhibitor such as polysaccharide and digestive enzyme RNase is introduced into saliva sample.
  • Japanese Unexamined Patent Publication No. 2012-24039 Japanese Unexamined Patent Publication No. 2017-023110 Japanese Unexamined Patent Publication No. 2016-182112
  • the present invention has been made in the background of the problems of the prior art. That is, for example, from a sample containing a large amount of digestive enzymes such as saliva and contaminants, viral RNA, especially viral RNA with an envelope, especially coronavirus RNA, is 1-step RT without prior purification. -PCR enables simple, rapid, and highly sensitive detection.
  • RNA virus for example, coronavirus, particularly SARS-nCOV-2
  • SARS-nCOV-2 coronavirus
  • Item 1 A method for inspecting RNA virus in a sample, which comprises the following steps. (1) A step of preparing a mixed solution containing a sample in which RNA has not been purified, an anionic polymer, and a polar organic solvent. (2) Step of heating the mixed solution, (3) A step of adding a one-step RT-PCR reaction solution containing (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcription activity to the heated mixed solution. (4) A step of carrying out a one-step RT-PCR reaction after sealing the reaction vessel.
  • Item 3. Item 2. The inspection method according to Item 1 or 2, wherein in the step (1), the content of the anionic polymer in the mixed solution is 0.00001% or more. Item 4.
  • Item 6. The inspection method according to any one of Items 1 to 3, wherein in the step (1), the mixed solution contains substantially no surfactant.
  • Item 5. Item 6.
  • Item 6. Item 6. The inspection method according to any one of Items 1 to 5, wherein the heating condition in the step (2) is 70 ° C. for 1 second or more.
  • Samples are selected from the group consisting of feces, pharyngeal swabs, nasal swabs, sputum, lung aspirates, cerebrospinal fluid, mouthwash, saliva, tears, cultured cells, culture supernatants, and environmental wiping test samples.
  • Item 8. Item 6.
  • Item 9. Item 6.
  • RNA virus is an RNA virus having an envelope.
  • the enveloped RNA virus consists of flavivirus family virus; togavirus family virus; coronavirus family virus; orthomixovirus family virus; rabdovirus family virus; bunyavirus family virus; paramyxovirus family virus; and phyllovirus family virus.
  • Item 11. Item 6. The test method according to any one of Items 1 to 10, wherein the RNA virus having an envelope is a coronaviridae virus. Item 12. Item 11.
  • the inspection method according to Item 11 wherein the coronavirus family virus is SARS (severe acute respiratory syndrome) coronavirus, MERS (Middle East respiratory syndrome) coronavirus, and SARS-nCOV-2 coronavirus.
  • Item 13 Item 6.
  • Item 14. Item 13.
  • the polar organic solvent is selected from the group consisting of ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pyridine, triethylamine dimethylformamide, hexamethylphosphoric triamide, dimethyl sulfoxide, acetone, and acetonitrile.
  • Item 6 The inspection method according to any one of Items 1 to 14, wherein the test method is at least one thereof. Item 16.
  • the anionic polymer is a polymer obtained by polymerizing a monomer having at least one anionic functional group selected from the group consisting of a sulfonic acid group, a carboxyl group, a phosphoric acid group, a sulfuric acid group, and a phosphonic acid group.
  • Item 6 The inspection method according to any one of Items 1 to 15. Item 17.
  • Anionic polymers include polyinosic acid, polycitidilic acid, polyguanylic acid, polyadenylic acid, polydeoxyinosic acid, polydeoxycitidilic acid, polydeoxyguanyl acid, polydeoxyadenylic acid, carrageenan, heparin, chondroitin sulfate, keratane sulfate, hyaluronic acid.
  • Item 6. The method for testing a virus according to any one of Items 1 to 16, which is at least one anionic polymer selected from the group. Item 18. Item 6. The test method according to any one of Items 1 to 17, wherein the DNA polymerase is any one selected from the group consisting of Taq, Tth and variants thereof. Item 19.
  • Items 1 to 18 characterized in that the origin of the reverse transcriptase is one selected from the group consisting of Moloney murine leukemia virus (MMRV), avian myeloblastosis virus (AMV) and variants thereof.
  • MMRV Moloney murine leukemia virus
  • AMV avian myeloblastosis virus
  • the 1-step RT-PCR reaction solution in step (4) is a quaternary ammonium salt having a structure in which three methyl groups are added to an amino group in an amino acid (hereinafter referred to as "betaine-like quaternary ammonium"), bovine serum.
  • RNA viruses comprising a one-step RT-PCR reaction solution comprising an anionic polymer, a polar organic solvent, (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcriptase activity.
  • kit. Item 23.
  • Item 6. The virus testing kit according to any one of Items 22 to 24, further comprising a hybridization probe corresponding to the detection region of the RNA virus to be detected.
  • Item 26. Item 6.
  • the enveloped RNA virus consists of flavivirus family virus; togavirus family virus; coronavirus family virus; orthomixovirus family virus; rabdovirus family virus; bunyavirus family virus; paramyxovirus family virus; and phyllovirus family virus.
  • Item 28. Item 6. The virus testing kit according to any one of Items 26 or 27, wherein the RNA virus having an envelope is a coronavirus.
  • Item 29. Item 27 or 28 for testing a virus according to Item 27 or 28, wherein the coronavirus family virus is SARS (severe acute respiratory syndrome) coronavirus, MERS (Middle East respiratory syndrome) coronavirus, SARS-nCOV-2. kit.
  • Item 30 Item 6. The virus test kit according to any one of Items 22 to 25, wherein the RNA virus does not have an envelope.
  • RNA virus having no envelope is selected from the group consisting of astroviridae virus; caliciviridae virus; picornaviridae virus; hepeviridae virus; and leoviridae virus.
  • Virus test kit is selected from the group consisting of astroviridae virus; caliciviridae virus; picornaviridae virus; hepeviridae virus; and leoviridae virus.
  • a reagent containing a sample, an anionic polymer, and a polar organic solvent is mixed and then heat-treated without isolating or purifying the nucleic acid from the sample, and then added to the 1-step RT-PCR reaction solution.
  • the influence of contaminants such as RNase that may be contained in the sample that has not undergone the purification step can be highly reduced, and the presence or absence of RNA virus in the sample can be detected.
  • the inspection work becomes more efficient, so that the number of inspections can be increased and it also contributes to the prevention of infectious diseases.
  • the step of purifying the viral RNA is omitted and the work is simplified, the risk of contamination between samples can be reduced. As a result, the risk of false positives can be suppressed, and the accuracy of inspection work can be further improved.
  • the risk of infection to the worker can be reduced by reducing the number of work processes in which the worker handles the infectious sample.
  • a similarly excellent effect can be exhibited in a sample that may contain the SARS-nCOV-2 coronavirus generated in 2019.
  • blood, feces excretion stool, rectal stool
  • vomitus urine, sputum, lymph fluid, plasma, ejaculation fluid, lung aspirate, cerebrospinal fluid, pharyngeal swab, nasal swab, gargling.
  • It also enables highly sensitive detection of coronavirus from samples containing a large amount of contaminants such as biological samples containing liquid, saliva, and tears, environmentally wiped samples, and samples containing cultured cells or culture supernatant.
  • the present invention can also be used for life science research, clinical diagnosis, food hygiene inspection, environmental inspection and the like.
  • One aspect of the present invention is an inspection of RNA virus in a sample, in which a mixed solution containing the sample, an anionic polymer, and a polar organic solvent is prepared and then heat-treated without purifying the viral RNA from the sample.
  • a method for testing for the presence of RNA virus comprising adding a one-step RT-PCR reagent comprising reverse transcriptase and DNA polymerase, or DNA polymerase having reverse transcription activity.
  • the method for testing a virus in a sample of the present invention comprises at least the following steps: (1) A step of preparing a mixed solution containing a sample in which RNA has not been purified, an anionic polymer, and a polar organic solvent. (2) Step of heating the mixed solution (3) A step of adding a one-step RT-PCR reaction solution containing (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcription activity to the mixed solution after the heat treatment. (4) A step of carrying out a one-step RT-PCR reaction after sealing the reaction vessel. It is preferable that the steps (1) to (4) are performed in the same container.
  • steps (1) and (4) it is preferable not to transfer all or a part of the mixed solution to another container between the steps (1) and (4).
  • the entire amount of the mixed solution of steps (1) and (2) may be subjected to steps (3) and (4), or a part thereof may be transferred to another container to carry out steps (3) and (4). Is also good.
  • the RNA virus to be inspected in the present invention may be an RNA virus having an envelope derived from the lipid double membrane or an RNA virus having no envelope.
  • the present invention excels in the effect of allowing highly sensitive testing of enveloped RNA viruses from unpurified samples.
  • Encapsulated RNA viruses include flaviviridae viruses (eg, hepatitis C virus, Japanese encephalitis virus, decavirus, pig fever virus); Togaviridae virus (eg, ruin virus).
  • Coronavirus family virus eg, SARS coronavirus, MERS coronavirus, SARS-nCOV-2 coronavirus
  • Orthomixoviridae virus eg, influenza virus
  • Rabdoviridae virus eg, mad dog disease virus
  • Bunyavirus family virus eg, Crimea-Congo fever virus, Hunter virus
  • Paramyxovirus family virus eg, measles virus, human RS virus
  • Phyllovirus family virus eg, Ebola virus
  • coronavirus family virus and more preferably SARS coronavirus, MERS coronavirus, and SARS-nCOV-2 coronavirus. It is useful for detection, especially for the detection of SARS-nCOV-2 coronavirus (also called SARS-CoV-2).
  • non-enveloped RNA viruses also referred to as "non-enveloped RNA viruses”
  • non-enveloped RNA viruses include astroviridae viruses (eg, astroviruses).
  • Calisivirus family virus eg, sapovirus, norovirus
  • picornavirus family virus eg, hepatitis A virus, echovirus, enterovirus, coxsackie virus, poliovirus, rhinovirus
  • hepevirus family virus eg, E
  • Hepatitis virus eg, E
  • Leovirus family virus eg, Rotavirus
  • Sapovirus is useful for detecting rotavirus, more preferably is useful for detecting norovirus, rotavirus, and particularly useful for detecting norovirus.
  • Examples of the sample used in the present invention include pharyngeal swab, nasal swab, sputum, feces (excreted feces, rectal feces), vomit, saliva, etc., but are not particularly limited and are derived from a living body. It can be used for all things. In particular, it is useful for detection from feces, pharyngeal swabs, nasal swabs, sputum, lung aspirates, cerebrospinal fluid, mouthwash, saliva, tears, cultured cells, and culture supernatants.
  • samples contain a large amount of digestive enzymes such as proteases and nucleic acid degrading enzymes (RNase, DNase) as impurities, and feces contain PCR reaction inhibitors such as Escherichia coli-derived proteins and nucleic acids.
  • RNase nucleic acid degrading enzymes
  • feces contain PCR reaction inhibitors such as Escherichia coli-derived proteins and nucleic acids.
  • reaction solution components such as enzymes, primers and nucleic acid probes used for RT-PCR reaction are digested or inactivated by the influence of impurities contained in the sample, and the detection sensitivity is lowered. ..
  • RNA is subjected to prior heat treatment in a mixed solution containing an anionic polymer and a polar organic solvent without isolating and purifying RNA from these samples using a commercially available RNA purification kit. It is characterized by being exposed from the structure and used for an RT-PCR reaction.
  • the sample may be subjected to direct detection, or the sample may be suspended in water, saline or buffer in order to reduce the influence of impurities on the reaction and obtain more stable test results. There may be.
  • the supernatant may be used after centrifugation. Alternatively, filter filtration may be performed.
  • the buffer solution is not particularly limited, and examples thereof include Hanks buffer solution, Tris buffer solution, phosphate buffer solution, glycine buffer solution, HEPES buffer solution, and tricine buffer solution.
  • the sample may be a sample treated with a sputazyme enzyme solution, although not particularly limited.
  • sample in the present invention is a sample containing cultured cells or a culture supernatant. Separation culture using cells is effective for virus isolation. Since the virus is contained in the culture supernatant after separation culture and the cultured cells, it can be a sample in the present invention.
  • cell types used for isolation culture include MDCK cells, hCK cells, VeroE6 / TMPRSS2 cells, CHO cells, HEK-293 cells, BHK-21 cells, Sf9 cells and Sf21 cells.
  • the wiping test is not particularly limited, but is a sample obtained by wiping the relevant section or equipment with a cotton swab or the like, eluting into water or a buffer solution, and concentrating with polyethylene glycol (PEG) precipitate or the like. ..
  • PEG polyethylene glycol
  • wipes include kitchen utensils such as cutting boards, kitchen knives, towels, and tableware, refrigerator handles and toilets, bathroom door knobs, washrooms, kitchens, toilets, bathroom faucets, cookers' hands and fingers, and bathrooms. , Toilets, washbasins, handrails, living rooms and other facilities. Although it is not a wiping test, it can also be applied to a concentrated sample of a sewage sample as an environmental test.
  • the polarity refers to an electronic bias existing in a molecule, and a molecule in which the centers of positive and negative charges in the molecule do not match is referred to as a polar molecule.
  • a solvent composed of polar molecules is called a polar solvent.
  • the polar solvents by using a polar organic solvent composed of an organic compound, it is possible to destabilize the higher-order structure of biomolecules such as nucleic acids and proteins. By utilizing this property, it has the effect of weakening the hydrophobic bond of the structural protein of the virus and destabilizing the capsid structure.
  • the destabilizing effect of the capsid structure by the polar organic solvent on the virus differs depending on the type of virus. It is considered that this is because the strength of hydrophobic binding and the like differs depending on the capsid protein possessed by the virus and the nature of the envelope.
  • polar organic solvent examples include ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, triethylamine, dimethylformamide, hexamethylphosphoric triamide, dimethylsulfoxide, acetone, acetonitrile and ethanol.
  • Preferred are methanol, triethylamine, dimethyl sulfoxide and acetone. Further, it may be a mixed solution containing two or more of these polar organic solvents.
  • the lower limit concentration of the polar organic solvent as a denaturing agent for the capsid protein depends on the type of the polar organic solvent and other additives, but is not particularly limited as long as the concentration is such that the capsid protein is denatured.
  • the effective concentration of the polar organic solvent differs for each virus, but usually the effective concentration of the polar organic solvent with respect to the sample amount is 10% or more and less than 100%. It is more preferably 30% or more and 90% or less, and further preferably 50% or more and 85% or less.
  • the effective concentration of the polar organic solvent is increased by setting the content of the polar organic solvent in the mixed solution in the step (1) to 20% or more, preferably 25% or more, and more preferably 30% or more. It may be possible to achieve.
  • the upper limit of the content of the polar organic solvent in the mixed solution in the step (1) is not particularly limited as long as the effect of the present invention is obtained, but is, for example, 90% or less, preferably 80% or less, more preferably 75% or less. It is better to do it.
  • the polar organic solvent is also commonly known as a PCR inhibitor. Therefore, among the polar organic solvents, those having a small difference between the concentration required for protein denaturation and the allowable concentration to be brought into PCR are sequentially added to the polar organic solvent, the sample, and the 1-step RT-PCR reaction solution. By doing so, the detection operation can be easily carried out in the same container from the denaturation of the capsid protein to the one-step RT-PCR reaction without opening and closing the container in the middle.
  • Examples of such polar organic solvents are particularly preferably dimethyl sulfoxide.
  • the concentration of dimethyl sulfoxide brought into the reaction solution is 2%.
  • 2% dimethyl sulfoxide is an acceptable concentration for bringing in RT-PCR solution.
  • the amount of the polar organic solvent (dimethyl sulfoxide) that can be suitably used in the present invention can be easily calculated in the same manner as described above.
  • the polar organic solvent may be used in combination with one or more kinds of surfactants, reducing agents, chelating agents, and metal salts, or may be substantially free of these surfactants and the like.
  • the mixture in step (1) may be substantially free of surfactant.
  • the surfactant it is possible to detect RNA virus in a sample with high sensitivity even if the pretreatment is performed in a state where the surfactant is not substantially contained. Further, it is desirable that the surfactant is substantially free of the surfactant because it may inhibit the RT-PCR reaction depending on the type of the surfactant.
  • substantially free means that the surfactant is not contained at a concentration that allows nucleic acid extraction from the RNA virus.
  • the concentration of the surfactant in the mixed solution of step (1) is 0. It is 001% or less, preferably 0.0001% or less, more preferably 0.00001% or less, and it is particularly preferable that the mixed solution of the step (1) does not contain any surfactant.
  • the anionic polymer is a polymer formed by polymerization mainly containing anionic monomers.
  • the anionic polymer used in the present invention mainly polymerizes a monomer having at least one anionic functional group selected from the group consisting of a sulfonic acid group, a carboxyl group, a phosphoric acid group, a sulfuric acid group, and a phosphonic acid group.
  • the polymer is preferably obtained by polymerizing a sulfonic acid group as a monomer.
  • Nucleic acid molecules such as RNA and DNA are also anionic polymers.
  • Nucleic acid-degrading enzymes contained in the sample bind to nucleic acid molecules, which are anionic polymers, and perform digestion.
  • the effect of suppressing the digestion of the target nucleic acid molecule by adding an anionic polymer that is not digested by the nucleic acid degrading enzyme to the reaction solution system. Is expected to play.
  • the anionic polymer is not particularly limited as long as it exhibits the effects of the present invention, but representative examples thereof include nucleic acid polymers (polyinosic acid, polycitidilic acid, polyguanyl acid, polyadenyl acid, polydeoxyinosic acid, polydeoxycitidilic acid, etc.
  • the anionic polymer may be in the form of a salt.
  • a salt may be an alkali metal salt (sodium salt, potassium salt, etc.), an alkaline earth metal salt (calcium salt, magnesium salt, etc.), or a hydrate salt. It is preferably an alkali metal salt, more preferably a sodium salt or a potassium salt, and even more preferably a sodium salt.
  • the average molecular weight of the anionic polymer is not particularly limited as long as the effect of the present invention is obtained.
  • the average molecular weight means the weight average molecular weight.
  • the average molecular weight of the anionic polymer depends on the molecular weight and degree of polymerization of the monomer as a constituent unit, and is, for example, 1,000 or more, preferably 5,000 or more, still more preferably 10,000 or more, still more preferably 50, It may be 000 or more.
  • the upper limit of the average molecular weight of the anionic polymer is not particularly limited as long as the effect of the present invention is obtained, but for example, it is 5,000,000 or less, preferably 1,000,000 or less, more preferably 5,000,000 or less. It can be:
  • the content of the anionic polymer in the mixed solution of the step (1) is preferably 0.00001% (v / v%) or more, and preferably 0.0001% or more. More preferably, it is more preferably 0.001% or more.
  • the upper limit of the content of the anionic polymer in the step (1) is not particularly limited as long as the effect of the present invention is obtained, but is, for example, 0.5% or less, more preferably 0.1% or less, still more preferably. It can be 0.01% or less.
  • the content of the anionic polymer brought into the 1-step RT-PCR reaction solution of step (3) is not particularly limited as long as it does not inhibit the RT-PCR reaction, but may be, for example, 0.05% or less, and more specifically. It may be 0.01% or less, and may be, for example, 0.00001 to 0.001%.
  • the time from preparing the mixed solution in the step (1) to carrying out the step (2) is within 5 minutes. By shortening the time from the end of the step (1) to the start of the step (2) in this way, a more rapid RNA virus test becomes possible.
  • the time from preparing the mixed solution in the step (1) to carrying out the step (2) is not particularly limited, but is preferably 5 minutes or less, more preferably 4 minutes or less, and 3 minutes. It is more preferably less than or equal to, more preferably 2 minutes or less, and may be 1 minute or less.
  • the lower limit of the time from preparing the mixed solution in the step (1) to carrying out the step (2) is not particularly limited, but may be, for example, 10 seconds or longer, preferably 30 seconds or longer. In the present invention, even if the pretreatment time in the step (1) is such a short time, it is possible to induce the exposure of RNA contained in the envelope or capsid, and the detection of RNA by 1-step RT-PCR can be induced. Can be made possible.
  • the heating condition of the step (2) carried out by the RNA virus testing method of the present invention may be 70 ° C. or higher.
  • the effect of the present invention can be obtained even more effectively by preferably carrying out at 80 ° C. or higher, more preferably 90 ° C. or higher, for example, 95 ° C.
  • the upper limit of the heating conditions in the step (2) is not particularly limited, but may be, for example, 100 ° C.
  • the upper limit is not particularly limited as long as the effect of the present invention is achieved, but for example, if it is set to 10 minutes or less, rapid RNA virus testing becomes possible.
  • the heating condition in the step (2) may be 70 ° C. for 1 second or longer, and as another preferred embodiment, the heating condition may be 80 ° C. or 90 ° C. for 1 second or longer.
  • the work of purifying virus-derived RNA from a sample is complicated and causes an increase in working time.
  • transfer of the reaction vessel containing the virus-containing sample, centrifugation, and the like raise the risk of scattering of the virus and virus-derived RNA.
  • the spread of the virus threatens the safety and health of workers, and at the same time, it means pollution of the inspection work environment. Since the scattered RNA virus is aerosolized in the workplace, there is a problem of contamination risk of other samples being inspected at the same time and infection risk of workers. Therefore, the method of inspecting the presence or absence of a virus using RT-PCR without virus-derived RNA purification from a sample has more significance than simplification of work.
  • the one-step RT-PCR solution added to the mixture contains reverse transcriptase and DNA polymerase. It is preferable to use Tth DNA polymerase, Taq DNA polymerase, or the like, which is a DNA polymerase having both reverse transcriptase activity. More preferably, the use of two enzymes, the use of at least two enzymes, reverse transcriptase and DNA polymerase.
  • the origin of the reverse transcriptase contained in the 1-step RT-PCR reaction solution is not particularly limited as long as RNA can be converted into DNA, but is MMLV (Murine leukemia Virus) -RT, AMV-RT (Avian Myeloblastosis Virus), HIV. -RT, RAV2-RT, EIAV-RT, Carboxydothermus hydrogenoformam DNA polymerase and variants thereof are exemplified. Particularly preferred examples include MMLV-RT, AMV-RT, or variants thereof.
  • Examples of the DNA polymerase contained in the 1-step RT-PCR reaction solution include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT and variants thereof. , Not particularly limited. More preferably, the use of Taq, Tth or variants thereof. Particularly preferred is the use of Tth or a variant thereof.
  • the enzymatic activity of the DNA polymerase during the reverse transcription reaction can be achieved by introducing it into a DNA polymerase having a heat-unstable block group in combination with an anti-DNA polymerase antibody or by chemical modification. Is suppressed, and it is preferable that it can be applied to hot-start PCR.
  • the variant of DNA polymerase refers to, for example, 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, more preferably 98% or more, with respect to the amino acid sequence of the wild-type DNA polymerase from which it is derived. % Or more, preferably 99% or more of sequence identity, and has the activity of amplifying DNA like wild-type DNA polymerase and, if necessary, the activity of converting RNA into cDNA. ..
  • any means known in the art can be used as a method for calculating the identity of the amino acid sequence.
  • the mutant that can be used in the present invention has one or several amino acids substituted, deleted, inserted and / or added in the amino acid sequence of the wild-type DNA polymerase from which it is derived (hereinafter, these are collectively referred to as "these".
  • polypeptide consisting of an amino acid sequence (also referred to as "mutation"), and may have an activity of converting RNA into DNA and an activity of amplifying DNA as in the case of wild-type DNA polymerase.
  • 1 or several pieces are, for example, 1 to 80 pieces, preferably 1 to 40 pieces, more preferably 1 to 10 pieces, still more preferably 1 to 5 pieces, still more preferably 1 to 3 pieces. It is possible, but not particularly limited.
  • the 1-step RT-PCR reaction solution used in the present invention contains reverse transcriptase and DNA polymerase, as well as a buffer, magnesium salt or manganese salt as an appropriate salt, deoxynucleotide triphosphate, and viral RNA to be detected. It may contain a primer pair corresponding to the detection target region, and may further contain an additive if necessary.
  • the buffer used in the present invention is not particularly limited, and examples thereof include Tris, Tricine, Bis-Tricine, and Bicine.
  • the pH was adjusted to 6 to 9, more preferably pH 7 to 8 with sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid or the like.
  • the concentration of the buffer to be added is 10 to 200 mM, more preferably 20 to 150 mM.
  • a salt solution is added in order to make the ionic conditions suitable for the reaction.
  • the salt solution include potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate and the like.
  • dNTP As the dNTP used in the present invention, dATP, dCTP, dGTP, and dTTP are added at 0.1 to 0.5 mM, respectively, and most commonly, about 0.2 mM is added. Prophylactic measures against cross-contamination may be taken by using dUTP as an alternative and / or as part of dTTP.
  • the magnesium salt include magnesium chloride, magnesium sulfate and magnesium acetate
  • manganese salt include manganese chloride, manganese sulfate and manganese acetate, and it is preferable to add about 1 to 10 mM.
  • a quaternary ammonium salt having a structure in which three methyl groups are added to an amino group in an amino acid (hereinafter referred to as “betaine-like quaternary ammonium”), It preferably comprises at least one selected from the group consisting of bovine serum albumin, glycerol, glycol and gelatin.
  • betaine-like quaternary ammonium salt examples include betaine (trimethylglycine) and L-carnitine, but any quaternary ammonium salt having a structure in which three methyl groups are added to an amino group in an amino acid can be used. It is not particularly limited.
  • the structure of the betaine-like quaternary ammonium salt is a compound having both positive and negative charges that is stable in the molecule, and exhibits properties like a surfactant, and is considered to cause destabilization of the virus structure. Furthermore, it is known to promote nucleic acid amplification of DNA polymerase.
  • the preferred concentration of the betaine-like quaternary ammonium salt is 0.1 M to 2 M, more preferably 0.2 M to 1.2 M.
  • the bovine serum albumin contained in the one-step RT-PCR reaction solution is preferably at least 0.5 mg / ml or more, more preferably at least 1 mg / ml or more.
  • the concentration of bovine serum albumin is preferably 2 mg / ml or more, more preferably 3 mg / mg or more, and good detection is possible.
  • Gelatin contained in the one-step RT-PCR reaction solution is derived from the skin, bones and tendons of animals such as cows and pigs, or the scales and skins of fish, and is considered to contribute to the stabilization of PCR enzymes.
  • the concentration used is preferably such that it stabilizes PCR amplification but does not interfere with fluorescence detection. It is preferably 1 to 5%, more preferably 1 to 2%.
  • the origin of gelatin is not limited, but those derived from fish are preferable to those derived from cattle and pigs in that the jelly strength is low and the handling of the reaction solution is good.
  • Accelerators useful in the present invention include, for example, glycerol, polyols, protease inhibitors, single strand binding proteins (SSBs), T4 gene 32 proteins, tRNA, sulfur or acetic acid-containing compounds, dimethylsulfoxide (DMSO), glycerol, ethylene.
  • Glycerol Propylene Glycol, Trimethylene Glycol, Formamide, Acetamide, Betaine, Ectoin, Trehalose, Dextran, Polyvinylpyrrolidone (PVP), Tetramethylammonium Chloride (TMC), Tetramethylammonium Hydroxide (TMAH), Tetramethylammonium Acetate (TMAA) ), Polyethylene glycol, Triton X-100, Triton X-114, Tween 20, Nonidet P40, Briji58 and the like, but are not limited thereto.
  • EGTA Ethyleneglycol-bis (2-aminoethyl ether) -N, N, N', N'-tetraacetic acid
  • BAPTA a chelating agent
  • the one-step RT-PCR reaction solution further contains one or more primer pairs corresponding to the target region in the step (3).
  • the primer pair used in the present invention is a primer pair corresponding to the detection region (target region) of the RNA virus to be detected, and two types of primers in which one primer is complementary to the DNA extension product of the other primer. A pair of primers can be mentioned. Further, as another embodiment, so-called multiplex PCR in which two or more pairs of the above primers are contained can be mentioned.
  • the target nucleic acid consists of subtypes, it may contain degenerate primers.
  • coronavirus SARS-nCOV-2
  • SARS-nCOV-2 coronavirus
  • an example of a primer pair is the "Pathogen Detection Manual 2019-nCoV” published by the National Institute of Infectious Diseases. (SEQ ID NOs: 1, 2, 4, 5), "2019-Novel Coronavirus (2019-nCoV) Real-time rRT-pCR PanelPrimers and Probes” (SEQ ID NOs: 7, 8) announced by the American Center for Disease Control and Prevention. 10, 11, 13, 14) can be mentioned and can be suitably used in the present invention, but the present invention is not limited thereto.
  • nucleocapsid protein (N) region of SARS-nCOV-2 by SEQ ID NOs: 1 and 2, SEQ ID NOs: 4 and 5, SEQ ID NOs: 7 and 8, SEQ ID NOs: 10 and 11, and SEQ ID NOs: 13 and 14. Is detected.
  • nucleocapsid (N) region In the detection of coronaviruses such as SARS-nCOV-2, nucleocapsid (N) region, envelope protein (E) region, spike protein (S) region, RNA-dependent RNA polymerase (RdRp) region, Open Reading Frame. Genes such as the (ORF) region can be detected, but the detection is not limited thereto.
  • the concentration of the primer to be used it is preferable that the concentration of the forward primer is 0.1 ⁇ M or more and 3 ⁇ M or less and the concentration of the reverse primer is 0.1 ⁇ M or more and 3 ⁇ M or less with respect to the entire RT-PCR reaction solution. .. More preferably, the concentration of the forward primer is 0.1 ⁇ M or more and 2 ⁇ M or less, and the concentration of the reverse primer is 0.5 ⁇ M or more and 2 ⁇ M or less.
  • Another aspect of the invention is a detection method comprising at least one labeled hybridization probe or double-stranded DNA-bound fluorescent compound.
  • Examples of the double-stranded DNA-bound fluorescent compound include SYBR (registered trademark) Green I, SYBR (registered trademark) Gold, SYTO-9, SYTP-13, SYTO-82 (Life Technologies), and EvaGreen (registered trademark; Biotium). , LCGreen (Idaho), LightCycler (registered trademark) 480 ResoLight (Roche Applied Science) and the like.
  • hybridization probe used in the present invention examples include TaqMan hydrolysis probe (US Pat. No. 5,210,015, US Pat. No. 5,538,848, US Pat. No. 5,487,972). , US Pat. No. 5,804,375), Molecular Beacon (US Pat. No. 5,118,801), FRET Hybridization Probe (International Publication No. 97/46707, International Publication No. 97/46712) , International Publication No. 97/46714 pamphlet) and the like.
  • coronavirus As the base sequence of the probe for detecting coronavirus (SARS-nCOV-2), which is one of the enveloped RNA viruses, "2019-Novel Coronavirus (2019-nCoV) Real-time RT-" announced by the American Center for Disease Control and Prevention The sequences (SEQ ID NOs: 3, 6) described in the “pCR PanelPrimers and Probes” (SEQ ID NOs: 9, 12, 15) and the "Pathogen Detection Manual 2019-nCoV” published by the National Institute of Infectious Diseases are mentioned. However, the present invention is not limited to this.
  • the probe sequence described above detects the N region of SARS-nCOV-2.
  • the target nucleic acid consists of subtypes, it may contain degenerate sequences.
  • the concentration of the fluorescently labeled probe is preferably 0.01 ⁇ M or more and 1.0 ⁇ M or less. More preferably, it is 0.013 ⁇ M or more and 0.75 ⁇ M or less, and even more preferably 0.02 ⁇ M or more and 0.5 ⁇ M or less.
  • kits for testing viral RNA in a sample a pretreatment solution containing a polar organic solvent and an anionic polymer, as well as reverse transcriptase and DNA polymerase (or DNA having reverse transcription activity).
  • Polymerase a kit for testing enveloped RNA virus, which comprises a one-step RT-PCR reaction solution.
  • the virus testing kit of the present invention contains at least a polar organic solvent, a reagent containing an anionic polymer, a reverse transcriptase, a DNA polymerase, and a one-step RT-PCR reaction solution.
  • the virus test kit of the present invention may contain a reagent containing both a polar organic solvent and an anionic polymer, or may contain a polar organic solvent and an anionic polymer as separate reagents. May be. From the viewpoint of facilitating the inspection work, it is preferable that the reagent is provided as a reagent containing both a polar organic solvent and an anionic polymer, and in a suitable kit of the present invention, a reagent containing both of them is preferably provided. Provided in a including manner.
  • the one-step RT-PCR reaction solution preferably contains at least one of betaine-like quaternary ammonium salt, bovine serum albumin, glycerol, glycol and gelatin.
  • kit of the present invention is provided in an embodiment in which various components as described above are enclosed in the same container or enclosed in separate containers, for example, packed in one package and including information on how to use the kit. can do. By using the kit of the present invention, it becomes possible to quickly and easily inspect the presence or absence of RNA virus in a sample.
  • Test example 1 Effect of anionic polymers on RT-PCR inhibition by RNase (1)
  • reaction solution having the composition shown below as the basic composition
  • coronavirus RNA in the reaction solution in the presence of RNase A was detected by 1-step RT-PCR.
  • SARS-CoV-2 Detection Kit -N1 set- As the detection reagent, SARS-CoV-2 Detection Kit -N1 set- (Toyobo) was used except for the pretreatment liquid.
  • the primer probe for detection which is an accessory of this reagent, is "2019-Novell Coronavirus (2019-nCoV) Real-time RT-PCR Panel Primers and Probes" (Effectives) published by the Centers for Disease Control and Prevention (CDC).
  • the probe used was FAM as a fluorescent label and BHQ1 (Black hole quencher) modified as a quenching group.
  • RT-PCR reaction solution 43 ⁇ L
  • Reaction solution 30 ⁇ L
  • Enzyme solution 5 ⁇ L
  • Primer / probe solution 5 ⁇ L
  • RNase free water 3 ⁇ L
  • VSA polyvinylsulfonate
  • RNA Control (Thermo Fisher Scientific) was mixed so as to have a final concentration of 50 copies / reaction to 5 copies / reaction, and 7 ⁇ L of the mixed solution was immediately added to 95 in a thermal cycler. Heat treatment was performed at ° C for 5 minutes.
  • RT-PCR reaction conditions Using StepOne plus (Thermo Fisher Scientific), a real-time PCR reaction was carried out in the following temperature cycle.
  • Test example 2 Examination of the effect of anionic polymer using inactivated virus Using the reaction solution having the composition shown below as the basic composition, inactivated coronavirus in the reaction solution in the presence of RNase A was detected by 1-step RT-PCR.
  • SARS-CoV-2 Detection Kit -N1 set- SARS-CoV-2 Detection Kit -N1 set- (Toyobo) was used except for the pretreatment liquid.
  • the primer probe for detection which is an accessory of this reagent, is "2019-Novell Coronavirus (2019-nCoV) Real-time RT-PCR Panel Primers and Probes" (Effectives) published by the Centers for Disease Control and Prevention (CDC).
  • the probe used was FAM as a fluorescent label and BHQ1 (Black hole quencher) modified as a quenching group.
  • RT-PCR reaction solution (41 ⁇ L) Reaction solution: 30 ⁇ L Enzyme solution: 5 ⁇ L Primer / probe solution: 5 ⁇ L RNase free water: 1 ⁇ L
  • Enzyme solution 5 ⁇ L
  • Primer / probe solution 5 ⁇ L
  • RNase free water 1 ⁇ L
  • Addition and pretreatment of inactivated virus and RNase A Add 1 ⁇ L of RNAse free water or 100 ng / ⁇ L RNase A (Nacalai Tesque) to 3 ⁇ L of 100% dimethyl sulfoxide, and add RNAse free water or sodium polyvinylsulfonate (PVSA) to the final concentration of 0.001%. 1 ⁇ L was mixed.
  • PVSA polyvinylsulfonate
  • Test example 3 Examination using saliva samples Using the reaction solution having the composition shown below as the basic composition, inactivated coronavirus in the reaction solution in the presence of a saliva sample was detected by 1-step RT-PCR.
  • SARS-CoV-2 Detection Kit -N1 set- As the detection reagent, SARS-CoV-2 Detection Kit -N1 set- (Toyobo) was used except for the pretreatment liquid.
  • the primer probe for detection which is an accessory of this reagent, is "2019-Novel Coronavirus (2019-nCoV) Real-time RT-PCR Panel Primers and Probes" (Effectives) published by the Centers for Disease Control and Prevention (CDC). : 24 Jan 2020), the probe used was FAM as a fluorescent label and BHQ1 (Black hole quencher) modified as a quenching group.
  • the present invention is suitably used in molecular biology research, and in tests for the purpose of clinical tests, food hygiene control, and the like.

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Abstract

The present invention provides a method and a kit for making it possible to detect the presence/absence of virus RNA by one-step RT-PCR, in which the RNA is not isolated from a specimen, and a sample that has been subjected to only a simple thermal pre-treatment is added to a reaction solution. In particular, provided is a means for detecting, at a high sensitivity, the presence of a coronavirus in a specimen. A method for testing the presence of an RNA virus according to one embodiment of the present invention comprises steps (1) to (4): (1) a step for preparing a liquid mixture that contains a specimen which has not been subjected to RNA purification, an anionic polymer, and a polar organic solvent; (2) a step for heating the liquid mixture; (3) a step for adding, to the liquid mixture which has been heated, a 1-step RT-PCR reaction solution that includes (i) a reverse transcriptase and a DNA polymerase or (ii) a DNA polymerase having reverse transcription activity; and (4) a step for performing 1-step RT-PCR reaction after tightly sealing the reaction container.

Description

改良されたウイルスの検出方法Improved virus detection method

　本発明は、核酸増幅によるＲＮＡウイルスの検出法に関する。より具体的には、試料から核酸の単離・精製をすることなく、試料とアニオン性ポリマーと極性有機溶媒とを含む混合液を調製後に熱処理を行い、リアルタイム逆転写ポリメラーゼ連鎖反応（ｑＲＴ－ＰＣＲ）の反応液を加えることによる、ＲＮＡウイルスの検出に関する。本発明により、例えば、唾液、咽頭ぬぐい液、鼻腔ぬぐい液、喀痰をはじめとする生体由来試料、糞便試料、血液試料、環境拭き取り試料等に含まれるＲＮＡウイルスを高感度にて検出することが可能である。本発明は、生命科学研究、臨床診断や食品衛生検査、環境検査等に利用できる。
The present invention relates to a method for detecting RNA virus by nucleic acid amplification. More specifically, without isolating and purifying nucleic acid from the sample, a mixed solution containing the sample, an anionic polymer, and a polar organic solvent is prepared and then heat-treated, and a real-time reverse transcription polymerase chain reaction (qRT-PCR) is performed. ) Is related to the detection of RNA virus by adding the reaction solution. INDUSTRIAL APPLICABILITY According to the present invention, for example, RNA virus contained in saliva, pharyngeal swab, nasal swab, biological sample such as sputum, fecal sample, blood sample, environmental wiping sample, etc. can be detected with high sensitivity. Is. The present invention can be used for life science research, clinical diagnosis, food hygiene inspection, environmental inspection, and the like.

　ウイルスはゲノム核酸としてデオキシリボ核酸を持つＤＮＡウイルスと、リボ核酸を持つＲＮＡウイルスに大別される。ウイルスは世代時間が短いため、突然変異率が高いことで知られているが、特にＲＮＡウイルスは変異しやすいとされている。このようなウイルスの変異は宿主への感染性や、感染した際の症状の種類や重篤度に大きな影響を与えることが知られている。そのため、変異したウイルスであっても迅速・的確に検出する手法を開発することは、感染症の拡大防止や封じ込めに重要である。
Viruses are roughly classified into DNA viruses having deoxyribonucleic acid as genomic nucleic acids and RNA viruses having ribonucleic acids. Viruses are known to have a high mutation rate due to their short generation time, but RNA viruses are particularly susceptible to mutation. It is known that such virus mutations have a great influence on the infectivity to the host and the type and severity of symptoms at the time of infection. Therefore, it is important to develop a method for promptly and accurately detecting even a mutated virus in order to prevent the spread of infectious diseases and to contain them.

　コロナウイルスは、風邪を含む呼吸器感染症引き起こす原因ウイルスであり、風邪の流行期において約１０～３５％程度はコロナウイルスが原因と言われている。変異型ウイルスが発生することも知られており、稀にＳＡＲＳ（重症急性呼吸器症候群）コロナウイルスやＭＥＲＳ（中東呼吸器症候群）コロナウイルス、新型コロナウイルス感染症（ＣＯＶＩＤ－１９）コロナウイルス（ＳＡＲＳ－ｎＣＯＶ－２）など致死性の重篤な呼吸器疾患を齎すものが発生することが知られている。したがって、コロナウイルスを簡便、迅速、高感度に検出することは、臨床診断、食品衛生検査、環境検査等で重要であることは言うまでもない。
Coronavirus is a causative virus that causes respiratory infections including colds, and it is said that about 10 to 35% of coronaviruses are caused by the coronavirus during the cold season. It is also known that mutant viruses occur, and rarely SARS (severe acute respiratory syndrome) coronavirus, MERS (Middle East respiratory syndrome) coronavirus, and new coronavirus infection (COVID-19) coronavirus (SARS). It is known that those that cause fatal and serious respiratory diseases such as -nCOV-2) occur. Therefore, it goes without saying that simple, rapid, and highly sensitive detection of coronavirus is important in clinical diagnosis, food hygiene inspection, environmental inspection, and the like.

　コロナウイルスの病原体検査では、電子顕微鏡法、ＥＬＩＳＡによる免疫学的抗原検出法、または核酸増幅技術を利用したウイルス遺伝子の検出法が開発されてきた。これらの検査法の中でも、高感度にコロナウイルスを検出可能な核酸増幅技術は、汎く使われている。コロナウイルスを核酸増幅法で検出するためにいくつかの技術が開発されてきた（例えば、非特許文献１、非特許文献２、特許文献１）。
In the pathogen test of coronavirus, a method for detecting a viral gene using electron microscopy, an immunological antigen detection method by ELISA, or a nucleic acid amplification technique has been developed. Among these test methods, nucleic acid amplification technology capable of detecting coronavirus with high sensitivity is widely used. Several techniques have been developed for detecting coronavirus by nucleic acid amplification method (for example, Non-Patent Document 1, Non-Patent Document 2, Patent Document 1).

　２０１９年に中国湖北省武漢市にて発生が確認された変異型コロナウイルスＳＡＲＳ－ｎＣＯＶ－２においては、ウイルスゲノムＲＮＡの解析が完了次第、核酸増幅技術を用いた検査方法が樹立された（例えば、非特許文献３、非特許文献４）。日本においても、国立感染症研究所の「病原体検出マニュアル２０１９－ｎＣｏＶ」にてＳＡＲＳ－ｎＣＯＶ－２の検出するための方法が記載されている（非特許文献５）。これらの手法において、試料中に含まれるコロナウイルスの検出には、試料からのウイルスＲＮＡの抽出および精製工程を伴う。ウイルスＲＮＡの抽出および精製工程、なかでも精製工程は煩雑であり、多くの作業時間を要していた。近年、インフルエンザウイルスの検出において、咽頭ぬぐい液試料を水溶性有機溶媒と界面活性剤が含まれる前処理液と混合したウイルス抽出液を試料とする方法が知られている（特許文献２、特許文献３）。また、Ｋ．Ｋａｎｇらは、高病原性北米産豚生殖器呼吸器症候群ウイルスＲＮＡを豚血清サンプルから直接ＲＴ－ＰＣＲにより検出できることを報告している（非特許文献６）。これらの手法では、ＲＮＡの抽出および精製工程を省略することで、試料中に含まれるＲＴ-ＰＣＲの反応阻害物質が反応液中に持ち込まれることになる。ＲＴ-ＰＣＲの反応阻害物質は試料の種類によって大きく異なる。例えば、唾液試料中では多糖類や消化酵素であるＲＮａｓｅなどのＰＣＲ反応阻害物質が多量に持ち込まれる。加えて、ウイルスの不活化およびＲＮＡの抽出条件はウイルス種によって大きく異なっていることも知られているが、先行技術文献においては、コロナウイルスに対する効果については全く言及がなされていない（特許文献２、特許文献３）。現在、コロナウイルス、特にＳＡＲＳ－ｎＣＯＶ－２コロナウイルスを含む咽頭・鼻腔ぬぐい液や唾液、喀痰、糞便試料などの生体試料やふき取り環境試料から、ＲＮＡの精製工程なく、１ステップＲＴ-ＰＣＲによって簡便・迅速に検出できる方法の開発が望まれている。
For the mutant coronavirus SARS-nCOV-2, which was confirmed to occur in Wuhan City, Hubei Province, China in 2019, a test method using nucleic acid amplification technology was established as soon as the analysis of viral genomic RNA was completed (for example). , Non-Patent Document 3, Non-Patent Document 4). Also in Japan, a method for detecting SARS-nCOV-2 is described in the "Pathogen Detection Manual 2019-nCoV" of the National Institute of Infectious Diseases (Non-Patent Document 5). In these techniques, detection of coronavirus contained in a sample involves extraction and purification of viral RNA from the sample. The extraction and purification process of viral RNA, especially the purification process, was complicated and required a lot of work time. In recent years, in the detection of influenza virus, a method has been known in which a virus extract obtained by mixing a pharyngeal swab sample with a pretreatment solution containing a water-soluble organic solvent and a surfactant is used as a sample (Patent Document 2, Patent Document). 3). In addition, K. Kang et al. Report that highly pathogenic North American pig genital respiratory syndrome viral RNA can be detected directly from pig serum samples by RT-PCR (Non-Patent Document 6). In these methods, by omitting the steps of RNA extraction and purification, the reaction inhibitor of RT-PCR contained in the sample is brought into the reaction solution. RT-PCR reaction inhibitors vary greatly depending on the type of sample. For example, a large amount of PCR reaction inhibitor such as polysaccharide and digestive enzyme RNase is introduced into saliva sample. In addition, it is known that the inactivation of the virus and the extraction conditions of RNA differ greatly depending on the virus species, but the prior art document does not mention the effect on the coronavirus at all (Patent Document 2). , Patent Document 3). Currently, it is easy to use 1-step RT-PCR from biological samples such as pharyngeal / nasal swabs containing coronavirus, especially SARS-nCOV-2 coronavirus, saliva, sputum, fecal samples, and wiping environment samples without the purification step of RNA. -It is desired to develop a method that can be detected quickly.

特開２０１２－２４０３９号公報Japanese Unexamined Patent Publication No. 2012-24039 特開２０１７－０２３１１０号公報Japanese Unexamined Patent Publication No. 2017-023110 特開２０１６－１８２１１２号公報Japanese Unexamined Patent Publication No. 2016-182112


　本発明は、かかる従来技術の課題を背景になされたものである。すなわち、例えば唾液などの消化酵素や夾雑物質を多量に含むような試料から、ウイルスＲＮＡ、特にエンベロープを持つウイルスのＲＮＡ、なかでもコロナウイルスのＲＮＡについて、事前に精製を行うことなく、１ステップＲＴ－ＰＣＲにより、簡便、迅速、高感度な検出を可能とすることである。
The present invention has been made in the background of the problems of the prior art. That is, for example, from a sample containing a large amount of digestive enzymes such as saliva and contaminants, viral RNA, especially viral RNA with an envelope, especially coronavirus RNA, is 1-step RT without prior purification. -PCR enables simple, rapid, and highly sensitive detection.

　本発明者らは、上記事情に鑑み、鋭意研究を行った結果、ウイルスＲＮＡの精製を行っていない試料において、アニオン性ポリマーと極性有機溶媒とを混合後、熱処理を行った後に、１ステップＲＴ－ＰＣＲに供することで、試料中に含まれ得るＲＮＡウイルス（例えば、コロナウイルス、特にＳＡＲＳ－ｎＣＯＶ－２）を検出できることを見出し、本発明に到達した。
In view of the above circumstances, the present inventors have conducted diligent research, and as a result, in a sample in which virus RNA has not been purified, an anionic polymer and a polar organic solvent are mixed, heat-treated, and then 1-step RT. -It has been found that RNA virus (for example, coronavirus, particularly SARS-nCOV-2) that can be contained in a sample can be detected by subjecting to PCR, and the present invention has been reached.

代表的な本願発明は、以下の通りである。

項１．試料中のＲＮＡウイルスの検査方法であって、以下の工程を含むことを特徴とする検査方法。

（１）ＲＮＡの精製を行っていない試料と、アニオン性ポリマーと、極性有機溶媒とを含む混合液を調製する工程、

（２）前記混合液を加熱する工程、

（３）加熱後の前記混合液に、（ｉ）逆転写酵素およびＤＮＡポリメラーゼまたは（ｉｉ）逆転写活性を有するＤＮＡポリメラーゼを含む１ステップＲＴ－ＰＣＲ反応液を添加する工程、

（４）反応容器を密閉後、１ステップＲＴ－ＰＣＲ反応を実施する工程。

項２．工程（１）において、混合液中の極性有機溶媒の含有率が２０％以上である項１に記載の検査方法。

項３．工程（１）において、混合液中のアニオン性ポリマーの含有率が０．００００１％以上である項１又は２に記載の検査方法。

項４．工程（１）において、混合液が界面活性剤を実質的に含まないことを特徴とする項１から項３のいずれかに記載の検査方法。

項５．工程（１）において混合液を調製後、工程（２）を実施するまでの時間が５分以内である項１から４のいずれかに記載の検査方法。

項６．工程（２）における加熱条件が、７０℃１秒以上であることを特徴とする項１から５のいずれかに記載の検査方法。

項７．試料が糞便、咽頭ぬぐい液、鼻腔ぬぐい液、喀痰、肺吸引物、脳脊髄液、うがい液、唾液、涙液、培養細胞、培養上清、及び環境中の拭き取り検査試料からなる群より選択される少なくとも１種である項１から６のいずれかに記載の検査方法。

項８．試料が水、生理食塩水、緩衝液若しくはスプタザイム酵素液に懸濁された懸濁液、又はそれらの遠心上清若しくは濃縮物である項１から７のいずれかに記載の検査方法。

項９．ＲＮＡウイルスがエンベロープを持つＲＮＡウイルスであることを特徴とする項１から項８のいずれかに記載の検査方法。

項１０．エンベロープを持つＲＮＡウイルスが、フラビウイルス科ウイルス；トガウイルス科ウイルス；コロナウイルス科ウイルス；オルトミクソウイルス科ウイルス；ラブドウイルス科ウイルス；ブニヤウイルス科ウイルス；パラミクソウイルス科ウイルス；及びフィロウイルス科ウイルスからなる群より選択されることを特徴とする項９に記載の検査方法。

項１１．エンベロープを持つＲＮＡウイルスがコロナウイルス科ウイルスである項１から１０のいずれかに記載の検査方法。

項１２．コロナウイルス科ウイルスがＳＡＲＳ（重症急性呼吸器症候群）コロナウイルス、ＭＥＲＳ（中東呼吸器症候群）コロナウイルス、ＳＡＲＳ－ｎＣＯＶ－２コロナウイルスである項１１に記載の検査方法。

項１３．ＲＮＡウイルスがエンベロープを持たないＲＮＡウイルスであることを特徴とする項１から項１２のいずれかに記載の検査方法。

項１４．エンベロープを持たないＲＮＡウイルスが、アストロウイルス科ウイルス；カリシウイルス科ウイルス；ピコルナウイルス科ウイルス；へペウイルス科ウイルス；及びレオウイルス科ウイルスからなる群より選択されることを特徴とする項１３に記載のウイルスの検査方法。

項１５．極性有機溶媒が、エタノール、メタノール、１－プロパノール、２－プロパノール、１－ブタノール、２－ブタノール、ピリジン、トリエチルアミンジメチルホルムアミド、ヘキサメチルホスホリックトリアミド、ジメチルスルホキシド、アセトン、およびアセトニトリルよりなる群から選択される少なくとも１種であることを特徴とする項１から１４のいずれかに記載の検査方法。

項１６．アニオン性ポリマーが、スルホン酸基、カルボキシル基、リン酸基、硫酸基、及びホスホン酸基からなる群より選択される少なくとも１種のアニオン性官能基を有するモノマーを重合して得られるポリマーである項１から項１５のいずれかに記載の検査方法。

項１７．アニオン性ポリマーが、ポリイノシン酸、ポリシチジル酸、ポリグアニル酸、ポリアデニル酸、ポリデオキシイノシン酸、ポリデオキシシチジル酸、ポリデオキシグアニル酸、ポリデオキシアデニル酸、カラギーナン、ヘパリン、コンドロイチン硫酸、ケラタン硫酸、ヒアルロン酸、ヘパラン硫酸、コンドロイチン、デルマタン硫酸、ポリビニルスルホン酸、ポリビニルホスホン酸、ポリスチレンスルホン酸、ポリアクリル酸、ポリアクリル酸／スルホン酸共重合体、ポリアクリル酸／マレイン酸共重合体およびこれらの塩からなる群から選択される少なくとも１種のアニオン性ポリマーである項１から１６のいずれかに記載のウイルスの検査方法。

項１８．ＤＮＡポリメラーゼが、Ｔａｑ、Ｔｔｈおよびそれらの変異体よりなる群から選択されるいずれかであることを特徴とする項１から１７のいずれかに記載の検査方法。

項１９．逆転写酵素の由来が、モロニーマウス白血病ウイルス（ＭＭＲＶ）、トリ骨髄芽球症ウイルス（ＡＭＶ）およびこれらの変異体からなる群より選択されるいずれかであることを特徴とする項１から１８のいずれかに記載の検査方法。

項２０．工程（４）における１ステップＲＴ－ＰＣＲ反応液が、アミノ酸におけるアミノ基に３個のメチル基を付加した構造を有する第４級アンモニウム塩（以下、「ベタイン様４級アンモニウム」という）、ウシ血清アルブミン、グリセロール、グリコールおよびゼラチンよりなる群から選択された少なくとも１つをさらに含むことを特徴とする項１から１９のいずれかに記載の検査方法。

項２１．ベタイン様４級アンモニウム塩が、ベタインまたはＬ－カルニチンである項２０に記載のウイルスの検査方法。

項２２．アニオン性ポリマー、極性有機溶媒、（ｉ）逆転写酵素及びＤＮＡポリメラーゼ又は（ｉｉ）逆転写酵素活性を有するＤＮＡポリメラーゼを含む１ステップＲＴ－ＰＣＲ反応液を含むことを特徴とするＲＮＡウイルスの検査用キット。

項２３．ベタイン様４級アンモニウム塩、ウシ血清アルブミン、グリセロール、グリコールおよびゼラチンよりなる群から選択された少なくとも１つをさらに含む項２２に記載の検査用キット。

項２４．検出対象のＲＮＡウイルスの検出領域に対応するプライマー対をさらに含むことを特徴とする項２２または２３に記載のウイルスの検査用キット。

項２５．検出対象のＲＮＡウイルスの検出領域に対応するハイブリダイゼーションプローブをさらに含むことを特徴とする項２２から２４いずれかに記載のウイルスの検査用キット。

項２６． ＲＮＡウイルスがエンベロープを持つことを特徴とする項２２から２５のいずれかに記載のウイルスの検査キット。

項２７．エンベロープを持つＲＮＡウイルスが、フラビウイルス科ウイルス；トガウイルス科ウイルス；コロナウイルス科ウイルス；オルトミクソウイルス科ウイルス；ラブドウイルス科ウイルス；ブニヤウイルス科ウイルス；パラミクソウイルス科ウイルス；及びフィロウイルス科ウイルスからなる群より選択されることを特徴とする項２６に記載のウイルスの検査用キット。

項２８．エンベロープを持つＲＮＡウイルスがコロナウイルスである項２６又は２７のいずれかに記載のウイルスの検査用キット。

項２９．コロナウイルス科ウイルスがＳＡＲＳ（重症急性呼吸器症候群）コロナウイルス、ＭＥＲＳ（中東呼吸器症候群）コロナウイルス、ＳＡＲＳ－ｎＣＯＶ－２であることを特徴とする項２７又は項２８に記載のウイルスの検査用キット。

項３０． ＲＮＡウイルスがエンベロープを持たないことを特徴とする項２２から２５のいずれかに記載のウイルスの検査キット。

項３１．エンベロープを持たないＲＮＡウイルスが、アストロウイルス科ウイルス；カリシウイルス科ウイルス；ピコルナウイルス科ウイルス；へペウイルス科ウイルス；及びレオウイルス科ウイルスからなる群より選択されることを特徴とする項３０に記載のウイルスの検査用キット。
Typical inventions of the present application are as follows.

Item 1. A method for inspecting RNA virus in a sample, which comprises the following steps.

(1) A step of preparing a mixed solution containing a sample in which RNA has not been purified, an anionic polymer, and a polar organic solvent.

(2) Step of heating the mixed solution,

(3) A step of adding a one-step RT-PCR reaction solution containing (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcription activity to the heated mixed solution.

(4) A step of carrying out a one-step RT-PCR reaction after sealing the reaction vessel.

Item 2. Item 2. The inspection method according to Item 1, wherein in the step (1), the content of the polar organic solvent in the mixed solution is 20% or more.

Item 3. Item 2. The inspection method according to Item 1 or 2, wherein in the step (1), the content of the anionic polymer in the mixed solution is 0.00001% or more.

Item 4. Item 6. The inspection method according to any one of Items 1 to 3, wherein in the step (1), the mixed solution contains substantially no surfactant.

Item 5. Item 6. The inspection method according to any one of Items 1 to 4, wherein the time from preparing the mixed solution in the step (1) to carrying out the step (2) is within 5 minutes.

Item 6. Item 6. The inspection method according to any one of Items 1 to 5, wherein the heating condition in the step (2) is 70 ° C. for 1 second or more.

Item 7. Samples are selected from the group consisting of feces, pharyngeal swabs, nasal swabs, sputum, lung aspirates, cerebrospinal fluid, mouthwash, saliva, tears, cultured cells, culture supernatants, and environmental wiping test samples. The inspection method according to any one of Items 1 to 6, which is at least one kind.

Item 8. Item 6. The inspection method according to any one of Items 1 to 7, wherein the sample is a suspension suspended in water, physiological saline, a buffer solution or a sputazyme enzyme solution, or a centrifugal supernatant or a concentrate thereof.

Item 9. Item 6. The inspection method according to any one of Items 1 to 8, wherein the RNA virus is an RNA virus having an envelope.

Item 10. The enveloped RNA virus consists of flavivirus family virus; togavirus family virus; coronavirus family virus; orthomixovirus family virus; rabdovirus family virus; bunyavirus family virus; paramyxovirus family virus; and phyllovirus family virus. Item 9. The inspection method according to Item 9, wherein the virus is selected from a group.

Item 11. Item 6. The test method according to any one of Items 1 to 10, wherein the RNA virus having an envelope is a coronaviridae virus.

Item 12. Item 11. The inspection method according to Item 11, wherein the coronavirus family virus is SARS (severe acute respiratory syndrome) coronavirus, MERS (Middle East respiratory syndrome) coronavirus, and SARS-nCOV-2 coronavirus.

Item 13. Item 6. The inspection method according to any one of Items 1 to 12, wherein the RNA virus is an RNA virus having no envelope.

Item 14. Item 13. Item 3. How to check for viruses.

Item 15. The polar organic solvent is selected from the group consisting of ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pyridine, triethylamine dimethylformamide, hexamethylphosphoric triamide, dimethyl sulfoxide, acetone, and acetonitrile. Item 6. The inspection method according to any one of Items 1 to 14, wherein the test method is at least one thereof.

Item 16. The anionic polymer is a polymer obtained by polymerizing a monomer having at least one anionic functional group selected from the group consisting of a sulfonic acid group, a carboxyl group, a phosphoric acid group, a sulfuric acid group, and a phosphonic acid group. Item 6. The inspection method according to any one of Items 1 to 15.

Item 17. Anionic polymers include polyinosic acid, polycitidilic acid, polyguanylic acid, polyadenylic acid, polydeoxyinosic acid, polydeoxycitidilic acid, polydeoxyguanyl acid, polydeoxyadenylic acid, carrageenan, heparin, chondroitin sulfate, keratane sulfate, hyaluronic acid. , Heparan sulfate, chondroitin, dermatane sulfate, polyvinyl sulfonic acid, polyvinyl phosphonic acid, polystyrene sulfonic acid, polyacrylic acid, polyacrylic acid / sulfonic acid copolymer, polyacrylic acid / maleic acid copolymer and salts thereof. Item 6. The method for testing a virus according to any one of Items 1 to 16, which is at least one anionic polymer selected from the group.

Item 18. Item 6. The test method according to any one of Items 1 to 17, wherein the DNA polymerase is any one selected from the group consisting of Taq, Tth and variants thereof.

Item 19. Items 1 to 18 characterized in that the origin of the reverse transcriptase is one selected from the group consisting of Moloney murine leukemia virus (MMRV), avian myeloblastosis virus (AMV) and variants thereof. The inspection method described in either.

Item 20. The 1-step RT-PCR reaction solution in step (4) is a quaternary ammonium salt having a structure in which three methyl groups are added to an amino group in an amino acid (hereinafter referred to as "betaine-like quaternary ammonium"), bovine serum. Item 6. The test method according to any one of Items 1 to 19, further comprising at least one selected from the group consisting of albumin, glycerol, glycol and gelatin.

Item 21. Item 20. The method for testing a virus according to Item 20, wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine.

Item 22. For testing RNA viruses comprising a one-step RT-PCR reaction solution comprising an anionic polymer, a polar organic solvent, (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcriptase activity. kit.

Item 23. Item 22. The test kit according to Item 22, further comprising at least one selected from the group consisting of betaine-like quaternary ammonium salt, bovine serum albumin, glycerol, glycol and gelatin.

Item 24. Item 22. The virus testing kit according to Item 22 or 23, further comprising a primer pair corresponding to the detection region of the RNA virus to be detected.

Item 25. Item 6. The virus testing kit according to any one of Items 22 to 24, further comprising a hybridization probe corresponding to the detection region of the RNA virus to be detected.

Item 26. Item 6. The virus test kit according to any one of Items 22 to 25, wherein the RNA virus has an envelope.

Item 27. The enveloped RNA virus consists of flavivirus family virus; togavirus family virus; coronavirus family virus; orthomixovirus family virus; rabdovirus family virus; bunyavirus family virus; paramyxovirus family virus; and phyllovirus family virus. Item 28. The virus testing kit according to Item 26, which is selected from the group.

Item 28. Item 6. The virus testing kit according to any one of Items 26 or 27, wherein the RNA virus having an envelope is a coronavirus.

Item 29. Item 27 or 28 for testing a virus according to Item 27 or 28, wherein the coronavirus family virus is SARS (severe acute respiratory syndrome) coronavirus, MERS (Middle East respiratory syndrome) coronavirus, SARS-nCOV-2. kit.

Item 30. Item 6. The virus test kit according to any one of Items 22 to 25, wherein the RNA virus does not have an envelope.

Item 31. Item 30, wherein the RNA virus having no envelope is selected from the group consisting of astroviridae virus; caliciviridae virus; picornaviridae virus; hepeviridae virus; and leoviridae virus. Virus test kit.

　本発明によって、試料から核酸の単離・精製をすることなく、試料と、アニオン性ポリマーと、極性有機溶媒とを含有する試薬とを混合後に熱処理を行い、１ステップＲＴ－ＰＣＲ反応液に添加するだけで、精製工程を経ていない試料中に含まれ得るＲＮａｓｅ等の夾雑物質による影響を高度に低減でき、試料中のＲＮＡウイルスの有無を検出することが可能になる。この結果、検査業務がさらに効率化することから、検査数を増やすことができ、感染症予防にも寄与する。また、ウイルスＲＮＡの精製工程が省略化され作業が簡易になることで、サンプル間でのコンタミリスク等を低減することができる。これにより、偽陽性発生リスクも抑えることができ、検査業務の精度を更に高めることができる。また、作業者が感染性のあるサンプルを取り扱う作業工程が減少することで、作業者への感染リスクも低減することができる。
According to the present invention, a reagent containing a sample, an anionic polymer, and a polar organic solvent is mixed and then heat-treated without isolating or purifying the nucleic acid from the sample, and then added to the 1-step RT-PCR reaction solution. By simply doing so, the influence of contaminants such as RNase that may be contained in the sample that has not undergone the purification step can be highly reduced, and the presence or absence of RNA virus in the sample can be detected. As a result, the inspection work becomes more efficient, so that the number of inspections can be increased and it also contributes to the prevention of infectious diseases. Further, since the step of purifying the viral RNA is omitted and the work is simplified, the risk of contamination between samples can be reduced. As a result, the risk of false positives can be suppressed, and the accuracy of inspection work can be further improved. In addition, the risk of infection to the worker can be reduced by reducing the number of work processes in which the worker handles the infectious sample.

　更に、本発明では２０１９年に発生したＳＡＲＳ－ｎＣＯＶ－２コロナウイルスを含みうる試料においても、同様に優れた効果が奏され得る。また、本発明によれば、血液、糞便（排泄便、直腸便）、嘔吐物、尿、痰、リンパ液、血漿、射精液、肺吸引物、脳脊髄液、咽頭拭い液、鼻腔拭い液、うがい液、唾液、涙液を含む生体由来試料、環境拭き取り試料、培養細胞または培養上清を含む試料等の夾雑物を多く含む試料からの、コロナウイルスの高感度の検出も可能とする。本発明は、生命科学研究、臨床診断や食品衛生検査、環境検査等にも利用できる。
Furthermore, in the present invention, a similarly excellent effect can be exhibited in a sample that may contain the SARS-nCOV-2 coronavirus generated in 2019. Further, according to the present invention, blood, feces (excretion stool, rectal stool), vomitus, urine, sputum, lymph fluid, plasma, ejaculation fluid, lung aspirate, cerebrospinal fluid, pharyngeal swab, nasal swab, gargling. It also enables highly sensitive detection of coronavirus from samples containing a large amount of contaminants such as biological samples containing liquid, saliva, and tears, environmentally wiped samples, and samples containing cultured cells or culture supernatant. The present invention can also be used for life science research, clinical diagnosis, food hygiene inspection, environmental inspection and the like.

ＲＮａｓｅ存在下における、アニオン性ポリマーを含む極性有機溶媒を用いたウイルスＲＮＡの検出結果を示す図である。It is a figure which shows the detection result of the viral RNA using the polar organic solvent containing an anionic polymer in the presence of RNase. ＲＮａｓｅ存在下における、アニオン性ポリマーを含む極性有機溶媒を用いた不活化ウイルスの検出結果を示す図である。It is a figure which shows the detection result of the inactivated virus using the polar organic solvent containing an anionic polymer in the presence of RNase. 唾液検体存在下における、アニオン性ポリマーを含む極性有機溶媒を用いたウイルスＲＮＡの検出結果を示す図である。It is a figure which shows the detection result of the virus RNA using the polar organic solvent containing an anionic polymer in the presence of a saliva sample.

　以下、本発明の実施形態を示しつつ、本発明についてさらに詳説するが、本発明はこれらに限定されない。なお、本明細書において使用される用語は、特に言及しない限り、当該分野で通常用いられる意味で用いられることが理解されるべきである。

　また、本明細書中に記載された非特許文献および特許文献の全てが、本明細書中において参考として援用される。本明細書中の「～」は「以上、以下」を意味し、例えば明細書中で「Ｘ～Ｙ」と記載されていれば「Ｘ以上、Ｙ以下」を示す。また本明細書中の「および／または」は、いずれか一方または両方を意味する。また本明細書において、単数形の表現は、他に言及しない限り、その複数形の概念をも含むことが理解されるべきである。
Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention, but the present invention is not limited thereto. It should be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise noted.

In addition, all of the non-patent documents and patent documents described in the present specification are incorporated herein by reference. "-" In the present specification means "greater than or equal to, less than or equal to", and for example, if "X to Y" is described in the present specification, it means "more than or equal to X, less than or equal to Y". In addition, "and / or" in the present specification means either one or both. It should also be understood herein that the singular representation also includes the concept of the plural, unless otherwise noted.

　本発明の一態様は、試料中のＲＮＡウイルスの検査であって、試料からウイルスＲＮＡの精製を実施することなく、試料とアニオン性ポリマーと極性有機溶媒とを含む混合液を調製後に熱処理を行い、逆転写酵素およびＤＮＡポリメラーゼ、または逆転写活性を有するＤＮＡポリメラーゼを含む１ステップＲＴ－ＰＣＲ試薬を添加することを包含する、ＲＮＡウイルスの存在の有無を検査するための方法である。
One aspect of the present invention is an inspection of RNA virus in a sample, in which a mixed solution containing the sample, an anionic polymer, and a polar organic solvent is prepared and then heat-treated without purifying the viral RNA from the sample. , A method for testing for the presence of RNA virus, comprising adding a one-step RT-PCR reagent comprising reverse transcriptase and DNA polymerase, or DNA polymerase having reverse transcription activity.

　好ましい実施形態では、本発明の試料中のウイルス検査方法は、少なくとも以下の工程が含むことを特徴とする：

（１）ＲＮＡの精製を行っていない試料と、アニオン性ポリマーと、極性有機溶媒とを含む混合液を調製する工程、

（２）前記混合液を加熱する工程

（３）前記熱処理後の混合液に、（ｉ）逆転写酵素およびＤＮＡポリメラーゼまたは（ｉｉ）逆転写活性を有するＤＮＡポリメラーゼを含む１ステップＲＴ－ＰＣＲ反応液を添加する工程、

（４）反応容器を密閉後、１ステップＲＴ－ＰＣＲ反応を実施する工程。

　前記工程（１）から（４）は、同一容器で行われることが好ましい。すなわち、工程（１）から（４）の間においては、混合液の全部または一部を別容器へ移し替えないことが好ましい。工程（１）および（２）の混合液の全量を工程（３）（４）に供してもよいし、その一部を別の容器に移し替えて工程（３）（４）を実施しても良い。
In a preferred embodiment, the method for testing a virus in a sample of the present invention comprises at least the following steps:

(1) A step of preparing a mixed solution containing a sample in which RNA has not been purified, an anionic polymer, and a polar organic solvent.

(2) Step of heating the mixed solution

(3) A step of adding a one-step RT-PCR reaction solution containing (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcription activity to the mixed solution after the heat treatment.

(4) A step of carrying out a one-step RT-PCR reaction after sealing the reaction vessel.

It is preferable that the steps (1) to (4) are performed in the same container. That is, it is preferable not to transfer all or a part of the mixed solution to another container between the steps (1) and (4). The entire amount of the mixed solution of steps (1) and (2) may be subjected to steps (3) and (4), or a part thereof may be transferred to another container to carry out steps (3) and (4). Is also good.

　本発明において検査対象となるＲＮＡウイルスは、脂質二重膜に由来するエンベロープを持つＲＮＡウイルスであっても、エンベロープを持たないＲＮＡウイルスであっても良い。特定の好ましい実施形態では、本発明はエンベロープを持つＲＮＡウイルスについて、未精製試料からの高感度な検査を可能にする効果に優れている。エンベロープを持つＲＮＡウイルス（「エンベロープＲＮＡウイルス」ともいう）としては、フラビウイルス科ウイルス（例えば、Ｃ型肝炎ウイルス、日本脳炎ウイルス、ジカウイルス、豚熱ウイルス）；トガウイルス科ウイルス（例えば、風疹ウイルス、チクングニアウイルス）；コロナウイルス科ウイルス（例えば、ＳＡＲＳコロナウイルス、ＭＥＲＳコロナウイルス、ＳＡＲＳ－ｎＣＯＶ－２コロナウイルス）；オルトミクソウイルス科ウイルス（例えば、インフルエンザウイルス）；ラブドウイルス科ウイルス（例えば、狂犬病ウイルス）；ブニヤウイルス科ウイルス（例えば、クリミヤ・コンゴ熱ウイルス、ハンタウイルス）；パラミクソウイルス科ウイルス（例えば、麻疹ウイルス、ヒトＲＳウイルス）；フィロウイルス科ウイルス（例えば、エボラウイルス）、などが挙げられるが、特に限定されるものではない。より一層確実に高い本発明の効果が得られ易いという観点から、好ましくは、コロナウイルス科ウイルスの検出に有用であり、より好ましくはＳＡＲＳコロナウイルス、ＭＥＲＳコロナウイルス、ＳＡＲＳ－ｎＣＯＶ－２コロナウイルスの検出に有用であり、なかでもＳＡＲＳ－ｎＣＯＶ－２コロナウイルス（ＳＡＲＳ－ＣｏＶ－２とも呼ばれる）の検出に有用である。
The RNA virus to be inspected in the present invention may be an RNA virus having an envelope derived from the lipid double membrane or an RNA virus having no envelope. In certain preferred embodiments, the present invention excels in the effect of allowing highly sensitive testing of enveloped RNA viruses from unpurified samples. Encapsulated RNA viruses (also referred to as "envelope RNA viruses") include flaviviridae viruses (eg, hepatitis C virus, Japanese encephalitis virus, decavirus, pig fever virus); Togaviridae virus (eg, ruin virus). , Chikungnia virus); Coronavirus family virus (eg, SARS coronavirus, MERS coronavirus, SARS-nCOV-2 coronavirus); Orthomixoviridae virus (eg, influenza virus); Rabdoviridae virus (eg, mad dog disease virus) ); Bunyavirus family virus (eg, Crimea-Congo fever virus, Hunter virus); Paramyxovirus family virus (eg, measles virus, human RS virus); Phyllovirus family virus (eg, Ebola virus), etc. , Is not particularly limited. From the viewpoint that a higher effect of the present invention can be obtained more reliably, it is preferably useful for detecting a coronavirus family virus, and more preferably SARS coronavirus, MERS coronavirus, and SARS-nCOV-2 coronavirus. It is useful for detection, especially for the detection of SARS-nCOV-2 coronavirus (also called SARS-CoV-2).

　本発明はエンベロープを持たないＲＮＡウイルス（「非エンベロープＲＮＡウイルス」ともいう）の検査にも利用することができ、このようなエンベロープを持たないＲＮＡウイルスとしては、アストロウイルス科ウイルス（例えば、アストロウイルス）；カリシウイルス科ウイルス（例えば、サポウイルス、ノロウイルス）；ピコルナウイルス科ウイルス（例えば、Ａ型肝炎ウイルス、エコーウイルス、エンテロウイルス、コクサッキーウイルス、ポリオウイルス、ライノウイルス）；へペウイルス科ウイルス（例えば、Ｅ型肝炎ウイルス）；レオウイルス科ウイルス（例えば、ロタウイルス）などが挙げられ、限定されるものではないが、好ましくはカリシウイルス科ウイルス及びレオウイルス科ウイルスの検出に有用であり、より好ましくはノロウイルス、サポウイルス、ロタウイルスの検出に有用であり、更に好ましくはノロウイルス、ロタウイルスの検出に有用であり、特にノロウイルスの検出に有用である。
The present invention can also be used for testing non-enveloped RNA viruses (also referred to as "non-enveloped RNA viruses"), and examples of such non-enveloped RNA viruses include astroviridae viruses (eg, astroviruses). ); Calisivirus family virus (eg, sapovirus, norovirus); picornavirus family virus (eg, hepatitis A virus, echovirus, enterovirus, coxsackie virus, poliovirus, rhinovirus); hepevirus family virus (eg, E) Hepatitis virus); Leovirus family virus (eg, Rotavirus) and the like, and are not limited, but are preferably useful for detecting Calisivir family virus and Leovirus family virus, and more preferably Norovirus. , Sapovirus, is useful for detecting rotavirus, more preferably is useful for detecting norovirus, rotavirus, and particularly useful for detecting norovirus.

　本発明において用いられる試料として、例えば咽頭ぬぐい液、鼻腔ぬぐい液、喀痰、糞便（排泄便、直腸便）、嘔吐物、唾液などが挙げられるが、特に限定されるものではなく、生体に由来するもの全般に用いることが可能である。特には、糞便、咽頭ぬぐい液、鼻腔ぬぐい液、喀痰、肺吸引物、脳脊髄液、うがい液、唾液、涙液、培養細胞、培養上清からの検出に有用である。これらの試料中には、夾雑物として、プロテアーゼ及び核酸分解酵素（ＲＮａｓｅ、ＤＮａｓｅ）等の消化酵素が多量に含まれている他、糞便には大腸菌由来のタンパク質及び核酸等のＰＣＲ反応阻害物質が多量含まれていることが特徴として挙げられる。ＲＴ－ＰＣＲ反応に用いる酵素やプライマー及び核酸プローブ等の反応液構成物は、試料中に含まれる夾雑物の影響により、消化または失活してしまい、検出感度が低下することが知られている。また、試料中に含まれる核酸分解酵素は、ウイルスから露出された核酸を消化してしまうことで、感度を低下させてしまったり、検出不可にして偽陰性の判定結果を生じさせるおそれがある。本発明においては、これら試料を市販のＲＮＡ精製キットでＲＮＡを単離・精製することなく、アニオン性ポリマーと極性有機溶媒とを含む混合液中にて、事前の熱処理を行うことでＲＮＡをウイルス構造から露出させ、ＲＴ－ＰＣＲ反応に用いることを特徴とするものである。前記試料は直接検出に供してもよいし、夾雑物の反応への影響を低減し、より安定した検査結果を得るために、水、生理食塩水または緩衝液に前記試料を懸濁した試料であってもよい。さらに、糞便など特に夾雑物の多い試料では、遠心分離し、その上清を使用してもよい。あるいは、フィルターろ過を実施してもよい。前記緩衝液としては、特に限定されるものではないが、ハンクス緩衝液、トリス緩衝液、リン酸緩衝液、グリシン緩衝液、ＨＥＰＥＳ緩衝液、トリシン緩衝液などが挙げられる。また、粘性の強い生体試料（例えば、粘性の強い喀痰を含む試料）の場合は、特に限定されないが、スプタザイム酵素液で処理した試料であってもよい。
Examples of the sample used in the present invention include pharyngeal swab, nasal swab, sputum, feces (excreted feces, rectal feces), vomit, saliva, etc., but are not particularly limited and are derived from a living body. It can be used for all things. In particular, it is useful for detection from feces, pharyngeal swabs, nasal swabs, sputum, lung aspirates, cerebrospinal fluid, mouthwash, saliva, tears, cultured cells, and culture supernatants. These samples contain a large amount of digestive enzymes such as proteases and nucleic acid degrading enzymes (RNase, DNase) as impurities, and feces contain PCR reaction inhibitors such as Escherichia coli-derived proteins and nucleic acids. One of the characteristics is that it is contained in a large amount. It is known that reaction solution components such as enzymes, primers and nucleic acid probes used for RT-PCR reaction are digested or inactivated by the influence of impurities contained in the sample, and the detection sensitivity is lowered. .. In addition, the nucleic acid-degrading enzyme contained in the sample may digest the nucleic acid exposed from the virus, thereby lowering the sensitivity or making it undetectable and producing a false-negative determination result. In the present invention, RNA is subjected to prior heat treatment in a mixed solution containing an anionic polymer and a polar organic solvent without isolating and purifying RNA from these samples using a commercially available RNA purification kit. It is characterized by being exposed from the structure and used for an RT-PCR reaction. The sample may be subjected to direct detection, or the sample may be suspended in water, saline or buffer in order to reduce the influence of impurities on the reaction and obtain more stable test results. There may be. Further, for a sample having a particularly large amount of contaminants such as feces, the supernatant may be used after centrifugation. Alternatively, filter filtration may be performed. The buffer solution is not particularly limited, and examples thereof include Hanks buffer solution, Tris buffer solution, phosphate buffer solution, glycine buffer solution, HEPES buffer solution, and tricine buffer solution. Further, in the case of a highly viscous biological sample (for example, a sample containing highly viscous sputum), the sample may be a sample treated with a sputazyme enzyme solution, although not particularly limited.

　本発明における別の態様の試料としては、培養細胞または培養上清を含む試料である。ウイルスの分離には、細胞を利用した分離培養が有効である。分離培養後の培養上清、および培養細胞中にはウイルスが含まれるため、本発明における試料となりうる。分離培養に利用される細胞の種類としては、ＭＤＣＫ細胞、ｈＣＫ細胞、ＶｅｒｏＥ６／ＴＭＰＲＳＳ2細胞、ＣＨＯ細胞、ＨＥＫ－２９３細胞、ＢＨＫ－２１ 細胞、Ｓｆ９ 細胞および　Ｓｆ２１細胞などが挙げられる。細胞を融解または破砕した際の細胞破砕液に、核酸分解酵素を多く含むことが知られているＵ９３７細胞等を含む試料に有効であるが、特に限定されるものではなく、これに準じる方法が広く含まれる。
Another example of the sample in the present invention is a sample containing cultured cells or a culture supernatant. Separation culture using cells is effective for virus isolation. Since the virus is contained in the culture supernatant after separation culture and the cultured cells, it can be a sample in the present invention. Examples of the cell types used for isolation culture include MDCK cells, hCK cells, VeroE6 / TMPRSS2 cells, CHO cells, HEK-293 cells, BHK-21 cells, Sf9 cells and Sf21 cells. It is effective for a sample containing U937 cells and the like, which are known to contain a large amount of nucleolytic enzyme in the cell disruption solution when the cells are thawed or disrupted, but the method is not particularly limited, and a method similar thereto is available. Widely included.

　本発明における別の態様の試料としては、環境中の拭き取り検査試料である。汚染経路の解明や施設環境等の汚染状況の把握には、ふき取り検査が有用である。本発明において、拭き取り検査とは、特に限定されるものでないが、例えば綿棒等で該当区画や設備等を拭き取り、水や緩衝液に溶出し、ポリエチレングリコール（ＰＥＧ）沈澱などで濃縮した試料である。具体的な拭き取り検査の要領としては、「ふきとり検体のノロウイルス検査法の改良」（ｈｔｔｐ：／／ｉｄｓｃ．ｎｉｈ．ｇｏ．ｊｐ／ｉａｓｒ／３２／３８２／ｄｊ３８２４．ｈｔｍｌ）などが例示されるが、特に限定はされるものではなく、これに準ずる方法が広く含まれる。拭き取り箇所の例としては、まな板や包丁、ふきん、食器などの調理器具類、冷蔵庫の取手やトイレ、浴室のドアノブ、洗面所、厨房、トイレ、浴室などの蛇口、調理者の手や指、浴室、トイレ、洗面、手すり、居室などの施設などが挙げられる。また、拭き取り検査ではないが、環境検査として、下水試料の濃縮試料にも適用できる。
Another aspect of the sample in the present invention is a wipe test sample in the environment. Wiping inspection is useful for clarifying the pollution route and grasping the pollution status such as the facility environment. In the present invention, the wiping test is not particularly limited, but is a sample obtained by wiping the relevant section or equipment with a cotton swab or the like, eluting into water or a buffer solution, and concentrating with polyethylene glycol (PEG) precipitate or the like. .. As a specific procedure for the wiping test, "improvement of the norovirus test method for the wiped sample" (http://idsc.nih.go.jp/iasr/32/382/dj3824.html) is exemplified. There is no particular limitation, and methods similar to this are widely included. Examples of wipes include kitchen utensils such as cutting boards, kitchen knives, towels, and tableware, refrigerator handles and toilets, bathroom door knobs, washrooms, kitchens, toilets, bathroom faucets, cookers' hands and fingers, and bathrooms. , Toilets, washbasins, handrails, living rooms and other facilities. Although it is not a wiping test, it can also be applied to a concentrated sample of a sewage sample as an environmental test.

　本発明において、極性とは分子内に存在する電子的な偏りを指し、分子内の正電荷と負電荷の重心が一致しない分子を極性分子という。極性分子により構成された溶媒を極性溶媒という。極性溶媒の中でも、有機化合物により構成された極性有機溶媒を用いることにより、核酸やタンパク質のような生体分子の高次構造を不安定化することができる。この性質を利用することで、ウイルスの構造タンパク質の疎水結合などを弱め、キャプシド構造を不安定化するような効果を奏するものである。上述した通り、ウイルスに対する極性有機溶媒によるキャプシド構造の不安定化効果は、ウイルスの種類によっても異なることが知られている。これはウイルスが保有するキャプシドタンパク質やエンベロープの性質の違いにより、疎水性結合などの強さが異なるからであると考えられる。
In the present invention, the polarity refers to an electronic bias existing in a molecule, and a molecule in which the centers of positive and negative charges in the molecule do not match is referred to as a polar molecule. A solvent composed of polar molecules is called a polar solvent. Among the polar solvents, by using a polar organic solvent composed of an organic compound, it is possible to destabilize the higher-order structure of biomolecules such as nucleic acids and proteins. By utilizing this property, it has the effect of weakening the hydrophobic bond of the structural protein of the virus and destabilizing the capsid structure. As described above, it is known that the destabilizing effect of the capsid structure by the polar organic solvent on the virus differs depending on the type of virus. It is considered that this is because the strength of hydrophobic binding and the like differs depending on the capsid protein possessed by the virus and the nature of the envelope.

　前記極性有機溶媒として、具体的にはエタノール、メタノール、１－プロパノール、２－プロパノール、１－ブタノール、２－ブタノール、トリエチルアミン、ジメチルホルムアミド、ヘキサメチルホスホリックトリアミド、ジメチルスルホキシド、アセトン、アセトニトリル、エタノール、メタノール、１－プロパノール、２－プロパノール、１－ブタノール、ピリジン等が挙げられるがこれに限られるものではない。好ましくは、メタノール、トリエチルアミン、ジメチルスルホキシド、アセトンである。また、これら極性有機溶媒を２つ以上含む混合溶液であっても良い。該極性有機溶媒のキャプシドタンパク質の変性剤としての下限の濃度としては、極性有機溶媒や他の添加剤の種類にもよるが、キャプシドタンパク質が変性される濃度であれば特に限定されるものではなく、また、ウイルスの種類によりキャプシドタンパク質やエンベロープが異なるため、極性有機溶媒の実効濃度は各ウイルス毎に異なっているが、通常は検体量に対する極性有機溶媒の実効濃度が１０％以上１００％未満、より好ましくは３０％以上９０％以下、さらに好ましくは５０％以上８５％以下である。例えば、前記工程（１）における混合液中の極性有機溶媒の含有率が２０％以上とすること、好ましくは２５％以上、より好ましくは３０％以上とすることにより上記極性有機溶媒の実効濃度を達成することが可能であり得る。前記工程（１）における混合液中の極性有機溶媒の含有率の上限は、本発明の効果を奏する限り特に限定されないが、例えば９０％以下、好ましくは８０％以下、より好ましくは７５％以下とするのがよい。なお、本発明において２種以上の極性有機溶媒を組み合わせて用いる場合は、それらの総量が上記含有率の範囲内になるようにして調整することが好ましい。
Specific examples of the polar organic solvent include ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, triethylamine, dimethylformamide, hexamethylphosphoric triamide, dimethylsulfoxide, acetone, acetonitrile and ethanol. , Methanol, 1-propanol, 2-propanol, 1-butanol, pyridine and the like, but are not limited thereto. Preferred are methanol, triethylamine, dimethyl sulfoxide and acetone. Further, it may be a mixed solution containing two or more of these polar organic solvents. The lower limit concentration of the polar organic solvent as a denaturing agent for the capsid protein depends on the type of the polar organic solvent and other additives, but is not particularly limited as long as the concentration is such that the capsid protein is denatured. In addition, since the capsid protein and envelope differ depending on the type of virus, the effective concentration of the polar organic solvent differs for each virus, but usually the effective concentration of the polar organic solvent with respect to the sample amount is 10% or more and less than 100%. It is more preferably 30% or more and 90% or less, and further preferably 50% or more and 85% or less. For example, the effective concentration of the polar organic solvent is increased by setting the content of the polar organic solvent in the mixed solution in the step (1) to 20% or more, preferably 25% or more, and more preferably 30% or more. It may be possible to achieve. The upper limit of the content of the polar organic solvent in the mixed solution in the step (1) is not particularly limited as long as the effect of the present invention is obtained, but is, for example, 90% or less, preferably 80% or less, more preferably 75% or less. It is better to do it. When two or more kinds of polar organic solvents are used in combination in the present invention, it is preferable to adjust the total amount so that the total amount thereof is within the above range.

　前記極性有機溶媒は、通常はＰＣＲの阻害剤としても知られる。そのため、前記極性有機溶媒の中でも、タンパク質の変性に必要な濃度とＰＣＲへの持込み許容濃度の相違が小さいものを選択することで、極性有機溶媒、試料、１ステップＲＴ－ＰＣＲ反応液と順次添加することで、キャプシドタンパク質の変性から１ステップＲＴ－ＰＣＲ反応まで同一容器で、途中で容器を開閉することなく簡便に検出操作が進められる。このような極性有機溶媒の例として、特に好ましくはジメチルスルホキシドが挙げられる。例えば、工程（１）でジメチルスルホキシド１μＬと試料１μＬを混合し、工程（３）で１ステップＲＴ－ＰＣＲ液４８μＬを加えた場合、反応液中に持ち込まれるジメチルスルホキシドの濃度は２％である。２％のジメチルスルホキシドはＲＴ－ＰＣＲ液の持ち込まれても許容される濃度である。本発明に好適に使用され得る極性有機溶媒（ジメチルスルホキシド）の量は、上記と同様にして容易に算出することができる。
The polar organic solvent is also commonly known as a PCR inhibitor. Therefore, among the polar organic solvents, those having a small difference between the concentration required for protein denaturation and the allowable concentration to be brought into PCR are sequentially added to the polar organic solvent, the sample, and the 1-step RT-PCR reaction solution. By doing so, the detection operation can be easily carried out in the same container from the denaturation of the capsid protein to the one-step RT-PCR reaction without opening and closing the container in the middle. Examples of such polar organic solvents are particularly preferably dimethyl sulfoxide. For example, when 1 μL of dimethyl sulfoxide and 1 μL of a sample are mixed in step (1) and 48 μL of 1-step RT-PCR solution is added in step (3), the concentration of dimethyl sulfoxide brought into the reaction solution is 2%. 2% dimethyl sulfoxide is an acceptable concentration for bringing in RT-PCR solution. The amount of the polar organic solvent (dimethyl sulfoxide) that can be suitably used in the present invention can be easily calculated in the same manner as described above.

　前記極性有機溶媒は、１種以上の界面活性剤、還元剤、キレート剤、金属塩と組み合わせて用いてもよいし、これらの界面活性剤などを実質的に含まないものであってもよい。特定の実施形態では、例えば、工程（１）における混合液が界面活性剤を実質的に含まないものであってもよい。本発明によれば、このように界面活性剤を実質的に含まない状態で前処理を行っても、試料中のＲＮＡウイルスを高感度に検出することが可能である。また界面活性剤は、その種類によってはＲＴ－ＰＣＲ反応を阻害する場合もあり得るため、界面活性剤を実質的に含まないことが望ましい。ここで実質的に含まないとは、ＲＮＡウイルスから核酸抽出し得るような濃度で界面活性剤を含まないことをいい、例えば、工程（１）の混合液中における界面活性剤の濃度が０．００１％以下、好ましくは０．０００１％以下、より好ましくは０．００００１％以下であり、なかでも、工程（１）の混合液が界面活性剤を全く含まないことが好ましい。
The polar organic solvent may be used in combination with one or more kinds of surfactants, reducing agents, chelating agents, and metal salts, or may be substantially free of these surfactants and the like. In a particular embodiment, for example, the mixture in step (1) may be substantially free of surfactant. According to the present invention, it is possible to detect RNA virus in a sample with high sensitivity even if the pretreatment is performed in a state where the surfactant is not substantially contained. Further, it is desirable that the surfactant is substantially free of the surfactant because it may inhibit the RT-PCR reaction depending on the type of the surfactant. Here, "substantially free" means that the surfactant is not contained at a concentration that allows nucleic acid extraction from the RNA virus. For example, the concentration of the surfactant in the mixed solution of step (1) is 0. It is 001% or less, preferably 0.0001% or less, more preferably 0.00001% or less, and it is particularly preferable that the mixed solution of the step (1) does not contain any surfactant.

　アニオン性ポリマーは、アニオン性モノマーを主とする重合によって形成されたポリマーである。例えば、本発明に用いるアニオン性ポリマーは、スルホン酸基、カルボキシル基、リン酸基、硫酸基、及びホスホン酸基からなる群より選択される少なくとも１種のアニオン性官能基を有するモノマーを主として重合して得られるポリマーであり、好ましくは、スルホン酸基をモノマーとして重合して得られるポリマーである。ＲＮＡやＤＮＡをはじめとする核酸分子もアニオン性ポリマーである。試料中に含まれる核酸分解酵素はアニオン性ポリマーである核酸分子と結合し、消化を行う。理論に束縛されることは望まないが、本発明においては、核酸分解酵素にて消化されることのないアニオン性ポリマーを反応液系に添加することで、標的とする核酸分子の消化を抑える効果を奏することが期待される。
The anionic polymer is a polymer formed by polymerization mainly containing anionic monomers. For example, the anionic polymer used in the present invention mainly polymerizes a monomer having at least one anionic functional group selected from the group consisting of a sulfonic acid group, a carboxyl group, a phosphoric acid group, a sulfuric acid group, and a phosphonic acid group. The polymer is preferably obtained by polymerizing a sulfonic acid group as a monomer. Nucleic acid molecules such as RNA and DNA are also anionic polymers. Nucleic acid-degrading enzymes contained in the sample bind to nucleic acid molecules, which are anionic polymers, and perform digestion. Although not bound by theory, in the present invention, the effect of suppressing the digestion of the target nucleic acid molecule by adding an anionic polymer that is not digested by the nucleic acid degrading enzyme to the reaction solution system. Is expected to play.

　前記アニオン性ポリマーとして、本発明の効果を奏する限り特に限定されないが、代表的なものとして、核酸ポリマー（ポリイノシン酸、ポリシチジル酸、ポリグアニル酸、ポリアデニル酸、ポリデオキシイノシン酸、ポリデオキシシチジル酸、ポリデオキシグアニル酸、ポリデオキシアデニル酸）、多糖類（カラギーナン、ヘパリン、コンドロイチン硫酸、ケラタン硫酸、ヒアルロン酸、ヘパラン硫酸、コンドロイチン、デルマタン硫酸）、ポリビニルスルホン酸、ポリビニルホスホン酸、ポリスチレンスルホン酸、ポリアクリル酸、ポリアクリル酸／スルホン酸共重合体、ポリアクリル酸／マレイン酸共重合体などが挙げられる。
The anionic polymer is not particularly limited as long as it exhibits the effects of the present invention, but representative examples thereof include nucleic acid polymers (polyinosic acid, polycitidilic acid, polyguanyl acid, polyadenyl acid, polydeoxyinosic acid, polydeoxycitidilic acid, etc. Polydeoxyguanyl acid, polydeoxyadenyl acid), polysaccharides (carrageenan, heparin, chondroitin sulfate, keratane sulfate, hyaluronic acid, heparan sulfate, chondroitin, dermatane sulfate), polyvinylsulfonic acid, polyvinylphosphonic acid, polystyrenesulfonic acid, polyacrylic acid Examples thereof include acids, polyacrylic acid / sulfonic acid copolymers, polyacrylic acid / maleic acid copolymers and the like.

　前記アニオン性ポリマーは、塩の形態であってもよい。例えば、アルカリ金属塩（ナトリウム塩、カリウム塩等）、アルカリ土類金属塩（カルシウム塩、マグネシウム塩）等であってもよく、水和物塩であってもよい。好ましくは、アルカリ金属塩であり、より好ましくはナトリウム塩、カリウム塩であり、更に好ましくはナトリウム塩であり得る。
The anionic polymer may be in the form of a salt. For example, it may be an alkali metal salt (sodium salt, potassium salt, etc.), an alkaline earth metal salt (calcium salt, magnesium salt, etc.), or a hydrate salt. It is preferably an alkali metal salt, more preferably a sodium salt or a potassium salt, and even more preferably a sodium salt.

　前記アニオン性ポリマーの平均分子量は、本発明の効果を奏する限り特に限定されない。なお、本明細書において平均分子量は重量平均分子量をいう。アニオン性ポリマーの平均分子量は、構成単位であるモノマーの分子量および重合度によるところがあるが、例えば１，０００以上、好ましくは５，０００以上、更に好ましくは１０，０００以上、更により好ましくは５０，０００以上であって良い。また、アニオン性ポリマーの平均分子量の上限値も本発明の効果を奏する限り特に限定されないが、例えば、５，０００，０００以下、好ましくは１，０００，０００以下、より好ましくは５，０００，０００以下であり得る。
The average molecular weight of the anionic polymer is not particularly limited as long as the effect of the present invention is obtained. In the present specification, the average molecular weight means the weight average molecular weight. The average molecular weight of the anionic polymer depends on the molecular weight and degree of polymerization of the monomer as a constituent unit, and is, for example, 1,000 or more, preferably 5,000 or more, still more preferably 10,000 or more, still more preferably 50, It may be 000 or more. Further, the upper limit of the average molecular weight of the anionic polymer is not particularly limited as long as the effect of the present invention is obtained, but for example, it is 5,000,000 or less, preferably 1,000,000 or less, more preferably 5,000,000 or less. It can be:

　特定の実施形態において、前記工程（１）の混合液中におけるアニオン性ポリマーの含有率は、０．００００１％（ｖ／ｖ％）以上であることが好ましく、０．０００１％以上であることがより好ましく、０．００１％以上であることが更に好ましい。前記工程（１）におけるアニオン性ポリマーの含有率の上限値は、本発明の効果を奏する限り特に限定されないが、例えば、０．５％以下、より好ましくは０．１％以下、更により好ましくは０．０１％以下とすることができる。このような濃度でアニオン性ポリマーを含有することにより、精製工程を経ていない試料中でウイルスから露出したＲＮＡが消化されてしまうのを効果的に防ぐことができ、及び／又は当該試料に含まれる夾雑物質等のＰＣＲ阻害物質による影響を効果的に防ぐことができ、結果としてウイルスＲＮＡの検出感度の低下を高度に抑制することができる。工程（３）の１ステップＲＴ－ＰＣＲ反応液に持ち込まれるアニオン性ポリマーの含有率は、ＲＴ－ＰＣＲ反応を阻害しない限り特に限定されないが、例えば、０．０５％以下であり得、更に具体的には０．０１％以下としてもよく、例えば、０．００００１～０．００１％としてもよい。
In a specific embodiment, the content of the anionic polymer in the mixed solution of the step (1) is preferably 0.00001% (v / v%) or more, and preferably 0.0001% or more. More preferably, it is more preferably 0.001% or more. The upper limit of the content of the anionic polymer in the step (1) is not particularly limited as long as the effect of the present invention is obtained, but is, for example, 0.5% or less, more preferably 0.1% or less, still more preferably. It can be 0.01% or less. By containing the anionic polymer at such a concentration, it is possible to effectively prevent the RNA exposed from the virus from being digested in the sample that has not undergone the purification step, and / or it is contained in the sample. It is possible to effectively prevent the influence of PCR inhibitors such as contaminants, and as a result, it is possible to highly suppress the decrease in the detection sensitivity of viral RNA. The content of the anionic polymer brought into the 1-step RT-PCR reaction solution of step (3) is not particularly limited as long as it does not inhibit the RT-PCR reaction, but may be, for example, 0.05% or less, and more specifically. It may be 0.01% or less, and may be, for example, 0.00001 to 0.001%.

　一つの実施形態において、本発明のＲＮＡウイルスの検査方法は、前記工程（１）において混合液を調製後、工程（２）を実施するまでの時間を５分以内とすることが好ましい。このように工程（１）終了後から工程（２）開始までの時間を短時間とすることで、より迅速なＲＮＡウイルスの検査が可能になる。前記工程（１）において混合液を調製後、工程（２）を実施するまでの時間は、特に限定されないが、５分以下であることが好ましく、４分以下であることがより好ましく、３分以下であることがより好ましく、２分以下であることが更に好ましく、１分以下であってもよい。前記工程（１）において混合液を調製後、工程（２）を実施するまでの時間の下限値は特に限定されないが、例えば、１０秒以上、好ましくは３０秒以上とすることができる。本発明では、工程（１）における前処理時間がこのように短時間であっても、エンベロープやキャプシドに内包されたＲＮＡの露出を誘導することができ、１ステップＲＴ－ＰＣＲでのＲＮＡの検出を可能にできる。
In one embodiment, in the method for testing RNA virus of the present invention, it is preferable that the time from preparing the mixed solution in the step (1) to carrying out the step (2) is within 5 minutes. By shortening the time from the end of the step (1) to the start of the step (2) in this way, a more rapid RNA virus test becomes possible. The time from preparing the mixed solution in the step (1) to carrying out the step (2) is not particularly limited, but is preferably 5 minutes or less, more preferably 4 minutes or less, and 3 minutes. It is more preferably less than or equal to, more preferably 2 minutes or less, and may be 1 minute or less. The lower limit of the time from preparing the mixed solution in the step (1) to carrying out the step (2) is not particularly limited, but may be, for example, 10 seconds or longer, preferably 30 seconds or longer. In the present invention, even if the pretreatment time in the step (1) is such a short time, it is possible to induce the exposure of RNA contained in the envelope or capsid, and the detection of RNA by 1-step RT-PCR can be induced. Can be made possible.

　一つの実施形態において、本発明のＲＮＡウイルス検査方法で実施する工程（２）の加熱条件は、７０℃以上であり得る。好ましくは、８０℃以上、より好ましくは９０℃以上、例えば９５℃で実施することにより、より一層効果的に本発明の効果を得ることができる。工程（２）の加熱条件の上限値は特に限定されないが、例えば１００℃であり得る。また、工程（２）の加熱条件は、上記加熱温度で１秒以上加熱することが好ましい。特定の好ましい実施形態では、３０秒以上加熱するのがよく、１分以上加熱するのがよく、３分以上加熱するのがよい。上限値は本発明の効果を奏する限り特に限定されないが、例えば１０分以下にすることで迅速なＲＮＡウイルスの検査が可能になる。例えば、一つの好ましい態様として、工程（２）における加熱条件は７０℃１秒以上としてもよいし、他の好ましい態様として８０℃又は９０℃で１秒以上の加熱条件としてもよい。
In one embodiment, the heating condition of the step (2) carried out by the RNA virus testing method of the present invention may be 70 ° C. or higher. The effect of the present invention can be obtained even more effectively by preferably carrying out at 80 ° C. or higher, more preferably 90 ° C. or higher, for example, 95 ° C. The upper limit of the heating conditions in the step (2) is not particularly limited, but may be, for example, 100 ° C. Further, as the heating condition of the step (2), it is preferable to heat at the above heating temperature for 1 second or longer. In certain preferred embodiments, heating is preferably 30 seconds or longer, 1 minute or longer, and 3 minutes or longer. The upper limit is not particularly limited as long as the effect of the present invention is achieved, but for example, if it is set to 10 minutes or less, rapid RNA virus testing becomes possible. For example, as one preferred embodiment, the heating condition in the step (2) may be 70 ° C. for 1 second or longer, and as another preferred embodiment, the heating condition may be 80 ° C. or 90 ° C. for 1 second or longer.

　検体からのウイルス由来ＲＮＡ精製作業は、煩雑であり作業時間を伸ばす原因となる。これに加えて、ウイルス含有検体の入った反応容器の移し替えや遠心分離作業等は、ウイルス及びウイルス由来ＲＮＡの飛散リスクが生じる。ウイルスの飛散は作業者の安全及び健康を脅かすものであると同時に、検査作業環境の汚染を意味する。飛散したＲＮＡウイルスは作業場においてエアロゾル化するため、同時に検査している他のサンプルの汚染リスクおよび作業者の感染リスクが問題となっている。このため、検体からのウイルス由来ＲＮＡ精製のないＲＴ－ＰＣＲを用いたウイルスの存在の有無を検査方法は、作業の単純化以上の意義を持っている。
The work of purifying virus-derived RNA from a sample is complicated and causes an increase in working time. In addition to this, transfer of the reaction vessel containing the virus-containing sample, centrifugation, and the like raise the risk of scattering of the virus and virus-derived RNA. The spread of the virus threatens the safety and health of workers, and at the same time, it means pollution of the inspection work environment. Since the scattered RNA virus is aerosolized in the workplace, there is a problem of contamination risk of other samples being inspected at the same time and infection risk of workers. Therefore, the method of inspecting the presence or absence of a virus using RT-PCR without virus-derived RNA purification from a sample has more significance than simplification of work.

　前記混合液に添加される１ステップＲＴ－ＰＣＲ溶液は、逆転写酵素およびＤＮＡポリメラーゼを含む。逆転写酵素活性を併せ持つＤＮＡポリメラーゼである、Ｔｔｈ　ＤＮＡポリメラーゼやＴａｑ　ＤＮＡポリメラーゼなどを使用することが好ましい。より好ましくは、二種の酵素の使用、逆転写酵素とＤＮＡポリメラーゼの少なくとも２種類の酵素の使用である。
The one-step RT-PCR solution added to the mixture contains reverse transcriptase and DNA polymerase. It is preferable to use Tth DNA polymerase, Taq DNA polymerase, or the like, which is a DNA polymerase having both reverse transcriptase activity. More preferably, the use of two enzymes, the use of at least two enzymes, reverse transcriptase and DNA polymerase.

　前記１ステップＲＴ－ＰＣＲ反応液に含まれる逆転写酵素の由来としては、ＲＮＡをＤＮＡに変換できれば特に限定されないが、ＭＭＬＶ（Ｍｏｌｏｎｅｙ　Ｍｕｒｉｎｅ　Ｌｅｕｋｅｍｉａ　Ｖｉｒｕｓ）－ＲＴ、ＡＭＶ－ＲＴ（Ａｖｉａｎ　Ｍｙｅｌｏｂｌａｓｔｏｓｉｓ　Ｖｉｒｕｓ）、ＨＩＶ－ＲＴ、ＲＡＶ２－ＲＴ、ＥＩＡＶ－ＲＴ、カルボキシドサーマス・ハイドロゲノフォルマン（Ｃａｒｂｏｘｙｄｏｔｈｅｒｍｕｓ　ｈｙｄｒｏｇｅｎｏｆｏｒｍａｍ）ＤＮＡポリメラーゼ）やその変異体が例示される。特に好ましい例としては、ＭＭＬＶ－ＲＴ、ＡＭＶ－ＲＴ、またはそれらの変異体が挙げられる。
The origin of the reverse transcriptase contained in the 1-step RT-PCR reaction solution is not particularly limited as long as RNA can be converted into DNA, but is MMLV (Murine leukemia Virus) -RT, AMV-RT (Avian Myeloblastosis Virus), HIV. -RT, RAV2-RT, EIAV-RT, Carboxydothermus hydrogenoformam DNA polymerase and variants thereof are exemplified. Particularly preferred examples include MMLV-RT, AMV-RT, or variants thereof.

　前記１ステップＲＴ－ＰＣＲ反応液に含まれるＤＮＡポリメラーゼとしては、Ｔａｑ、Ｔｔｈ，Ｂｓｔ，ＫＯＤ，Ｐｆｕ，Ｐｗｏ、Ｔｂｒ，Ｔｆｉ，Ｔｆｌ，Ｔｍａ，Ｔｎｅ、Ｖｅｎｔ，ＤＥＥＰＶＥＮＴやこれらの変異体が挙げられるが、特に限定されない。より好ましくは、Ｔａｑ、Ｔｔｈ又はこれらの変異体の使用である。特に好ましくはＴｔｈ又はその変異体の使用である。さらに、非特異的反応抑制の効果を高めるため、抗ＤＮＡポリメラーゼ抗体との併用、あるいは化学修飾により熱不安定ブロック基のＤＮＡポリメラーゼへ導入することで、逆転写反応の間、ＤＮＡポリメラーゼの酵素活性を抑制され、ホットスタートＰＣＲへの適用ができることが好ましい。
Examples of the DNA polymerase contained in the 1-step RT-PCR reaction solution include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT and variants thereof. , Not particularly limited. More preferably, the use of Taq, Tth or variants thereof. Particularly preferred is the use of Tth or a variant thereof. Furthermore, in order to enhance the effect of suppressing non-specific reactions, the enzymatic activity of the DNA polymerase during the reverse transcription reaction can be achieved by introducing it into a DNA polymerase having a heat-unstable block group in combination with an anti-DNA polymerase antibody or by chemical modification. Is suppressed, and it is preferable that it can be applied to hot-start PCR.

　本明細書において、ＤＮＡポリメラーゼの変異体とは、その由来である野生型ＤＮＡポリメラーゼのアミノ酸配列に対して、例えば８５％以上、好ましくは９０％以上、より好ましくは９５％以上、更に好ましくは９８％以上、なかでも好ましくは９９％以上の配列同一性を有し、且つ、野生型ＤＮＡポリメラーゼと同様にＤＮＡを増幅する活性、及び必要に応じてＲＮＡをｃＤＮＡに変換する活性を有するものをいう。ここで、アミノ酸配列の同一性を算出する方法としては、当該分野で公知の任意の手段で行うことができる。例えば、市販の又は電気通信回線（インターネット）を通じて利用可能な解析ツールを用いて算出することができ、一例として、全米バイオテクノロジー情報センター（ＮＣＢＩ）の相同性アルゴリズムＢＬＡＳＴ（Ｂａｓｉｃ　ｌｏｃａｌ　ａｌｉｇｎｍｅｎｔ　ｓｅａｒｃｈ　ｔｏｏｌ）ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ＢＬＡＳＴ／においてデフォルト（初期設定）のパラメータを用いることにより、アミノ酸配列の同一性を算出することが可能である。また、本発明に用いられ得る変異体は、その由来である野生型ＤＮＡポリメラーゼのアミノ酸配列において、１又は数個のアミノ酸が置換、欠失、挿入および／または付加（以下、これらを纏めて「変異」ともいう）したアミノ酸配列からなるポリペプチドであり、且つ、野生型ＤＮＡポリメラーゼと同様にＲＮＡをｃＤＮＡに変換する活性及びＤＮＡを増幅する活性を有するものであってもよい。ここで１又は数個とは、例えば、１～８０個、好ましくは１～４０個、よりこのましくは１～１０個、さらに好ましくは１～５個、更により好ましくは１～３個であり得るが、特に限定されない。
As used herein, the variant of DNA polymerase refers to, for example, 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, more preferably 98% or more, with respect to the amino acid sequence of the wild-type DNA polymerase from which it is derived. % Or more, preferably 99% or more of sequence identity, and has the activity of amplifying DNA like wild-type DNA polymerase and, if necessary, the activity of converting RNA into cDNA. .. Here, as a method for calculating the identity of the amino acid sequence, any means known in the art can be used. For example, it can be calculated using commercially available analysis tools available on the market or through telecommunications lines (Internet), for example, the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http. : // www. ncbi. nlm. nih. By using the default (initial setting) parameters in gov / BLAST /, it is possible to calculate the identity of the amino acid sequence. In addition, the mutant that can be used in the present invention has one or several amino acids substituted, deleted, inserted and / or added in the amino acid sequence of the wild-type DNA polymerase from which it is derived (hereinafter, these are collectively referred to as "these". It may be a polypeptide consisting of an amino acid sequence (also referred to as "mutation"), and may have an activity of converting RNA into DNA and an activity of amplifying DNA as in the case of wild-type DNA polymerase. Here, 1 or several pieces are, for example, 1 to 80 pieces, preferably 1 to 40 pieces, more preferably 1 to 10 pieces, still more preferably 1 to 5 pieces, still more preferably 1 to 3 pieces. It is possible, but not particularly limited.

　本発明に用いられる１ステップＲＴ－ＰＣＲ反応液には、逆転写酵素およびＤＮＡポリメラーゼの他、緩衝剤、適当な塩として、マグネシウム塩又はマンガン塩、デオキシヌクレオチド三リン酸、検出対象のウイルスＲＮＡの検出対象領域に対応するプライマー対を含み、さらに必要に応じて添加剤を含んでいてもよい。
The 1-step RT-PCR reaction solution used in the present invention contains reverse transcriptase and DNA polymerase, as well as a buffer, magnesium salt or manganese salt as an appropriate salt, deoxynucleotide triphosphate, and viral RNA to be detected. It may contain a primer pair corresponding to the detection target region, and may further contain an additive if necessary.

　本発明で使用される緩衝剤としては、特に限定されないが、トリス（Ｔｒｉｓ），トリシン（Ｔｒｉｃｉｎｅ），ビスートリシン（Ｂｉｓ－Ｔｒｉｃｉｎｅ），ビシン（Ｂｉｃｉｎｅ）などが挙げられる。硫酸、塩酸、酢酸、リン酸などでｐＨを６～９、より好ましくはｐＨ７～８に調整されたものである。また、添加する緩衝剤の濃度としては、１０～２００ｍＭ，より好ましくは２０～１５０ｍＭで使用される。この際、反応に適当なイオン条件とするために、塩溶液が加えられる。塩溶液としては、塩化カリウム、酢酸カリウム、硫酸カリウム、硫酸アンモニウム、塩化アンモニウム、酢酸アンモニウムなどが挙げられる。
The buffer used in the present invention is not particularly limited, and examples thereof include Tris, Tricine, Bis-Tricine, and Bicine. The pH was adjusted to 6 to 9, more preferably pH 7 to 8 with sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid or the like. The concentration of the buffer to be added is 10 to 200 mM, more preferably 20 to 150 mM. At this time, a salt solution is added in order to make the ionic conditions suitable for the reaction. Examples of the salt solution include potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate and the like.

　本発明で使用されるｄＮＴＰとしては、ｄＡＴＰ，ｄＣＴＰ，ｄＧＴＰ，ｄＴＴＰがそれぞれ０．１～０．５ｍＭ、最も一般的には０．２ｍＭ程度加えられる。ｄＴＴＰの代わり及び／又は一部としてｄＵＴＰを使用することによって、クロスコンタミネーションに対する予防処置をとってもよい。マグネシウム塩としては、塩化マグネシウム、硫酸マグネシウム、酢酸マグネシウム、マンガン塩としては、塩化マンガン、硫酸マンガン、酢酸マンガンなどが例示され、１～１０ｍＭ程度加えられることが好ましい。　
As the dNTP used in the present invention, dATP, dCTP, dGTP, and dTTP are added at 0.1 to 0.5 mM, respectively, and most commonly, about 0.2 mM is added. Prophylactic measures against cross-contamination may be taken by using dUTP as an alternative and / or as part of dTTP. Examples of the magnesium salt include magnesium chloride, magnesium sulfate and magnesium acetate, and examples of the manganese salt include manganese chloride, manganese sulfate and manganese acetate, and it is preferable to add about 1 to 10 mM.
さらに１ステップＲＴ－ＰＣＲ反応液に含まれる添加剤としては、アミノ酸におけるアミノ基に３個のメチル基を付加した構造を有する第４級アンモニウム塩（以下、「ベタイン様４級アンモニウム」という）、ウシ血清アルブミン、グリセロール、グリコールおよびゼラチンよりなる群から選択された少なくとも１つを含むことが好ましい。
Further, as an additive contained in the 1-step RT-PCR reaction solution, a quaternary ammonium salt having a structure in which three methyl groups are added to an amino group in an amino acid (hereinafter referred to as “betaine-like quaternary ammonium”), It preferably comprises at least one selected from the group consisting of bovine serum albumin, glycerol, glycol and gelatin.

　前記ベタイン様４級アンモニウム塩としては、ベタイン（トリメチルグリシン）、Ｌ－カルニチンなどが挙げられるが、アミノ酸におけるアミノ基に３個のメチル基を付加した構造を有する第４級アンモニウム塩であれば、特に限定されるものではない。ベタイン様４級アンモニウム塩が有する構造は分子内に安定な正、負の両電荷を持つ化合物で、界面活性剤のような性質を示し、ウイルス構造の不安定化を引き起こすものと考えられる。さらに、ＤＮＡポリメラーゼの核酸増幅を促進することが知られる。好ましい前記ベタイン様４級アンモニウム塩濃度は０．１Ｍ～２Ｍであり、より好ましくは０．２Ｍ～１．２Ｍである。
Examples of the betaine-like quaternary ammonium salt include betaine (trimethylglycine) and L-carnitine, but any quaternary ammonium salt having a structure in which three methyl groups are added to an amino group in an amino acid can be used. It is not particularly limited. The structure of the betaine-like quaternary ammonium salt is a compound having both positive and negative charges that is stable in the molecule, and exhibits properties like a surfactant, and is considered to cause destabilization of the virus structure. Furthermore, it is known to promote nucleic acid amplification of DNA polymerase. The preferred concentration of the betaine-like quaternary ammonium salt is 0.1 M to 2 M, more preferably 0.2 M to 1.2 M.

　前記１ステップＲＴ－ＰＣＲ反応液に含まれるウシ血清アルブミンとしては、好ましくは少なくとも０．５ｍｇ／ｍｌ以上、より好ましくは少なくとも１ｍｇ／ｍｌ以上である。夾雑物の多い試料では、ウシ血清アルブミンの濃度が好ましくは２ｍｇ／ｍｌ以上、さらに好ましくは３ｍｇ／ｍｇ以上で、良好な検出が可能となる。
The bovine serum albumin contained in the one-step RT-PCR reaction solution is preferably at least 0.5 mg / ml or more, more preferably at least 1 mg / ml or more. In a sample containing a large amount of impurities, the concentration of bovine serum albumin is preferably 2 mg / ml or more, more preferably 3 mg / mg or more, and good detection is possible.

　前記１ステップＲＴ－ＰＣＲ反応液に含まれるゼラチンは、ウシや豚などの動物の皮膚や骨、腱、あるいは魚の鱗や皮に由来し、ＰＣＲ酵素の安定化に寄与すると考えられている。使用濃度としては、ＰＣＲ増幅を安定化する一方で、蛍光検出を妨げない程度が好ましい。好ましくは１～５％、さらに好ましくは１～２％である。特にゼラチンの由来については限定されるものではないが、ウシや豚由来よりも魚由来のものの方が、ゼリー強度が低く、反応液のハンドリングがよい点で好ましい。
Gelatin contained in the one-step RT-PCR reaction solution is derived from the skin, bones and tendons of animals such as cows and pigs, or the scales and skins of fish, and is considered to contribute to the stabilization of PCR enzymes. The concentration used is preferably such that it stabilizes PCR amplification but does not interfere with fluorescence detection. It is preferably 1 to 5%, more preferably 1 to 2%. In particular, the origin of gelatin is not limited, but those derived from fish are preferable to those derived from cattle and pigs in that the jelly strength is low and the handling of the reaction solution is good.

　さらには、当該技術分野でＲＴ－ＰＣＲを促進することが知られる物質と組み合わせて使用することもできる。本発明において有用な促進物質とは、例えば、グリセロール、ポリオール、プロテアーゼインヒビター、シングルストランド結合タンパク質（ＳＳＢ）、Ｔ４遺伝子３２タンパク質、ｔＲＮＡ、硫黄または酢酸含有化合物類、ジメチルスルホキシド（ＤＭＳＯ）、グリセロール、エチレングリコール、プロピレングリコール、トリメチレングリコール、ホルムアミド、アセトアミド、ベタイン、エクトイン、トレハロース、デキストラン、ポリビニルピロリドン（ＰＶＰ），塩化テトラメチルアンモニウム（ＴＭＡＣ）、水酸化テトラメチルアンモニウム（ＴＭＡＨ）、酢酸テトラメチルアンモニウム（ＴＭＡＡ）、ポリエチレングリコール、トリトンＸ－１００（ＴｒｉｔｏｎＸ－１００）、トリトンＸ－１１４（ＴｒｉｔｏｎＸ－１１４）、ツイーン２０（Ｔｗｅｅｎ２０），ノニデットＰ４０、Ｂｒｉｊｉ５８などが挙げられるが、これらに限定されない。さらに反応阻害を低減するように、エチレングリコール-ビス(２－アミノエチルエーテル）－Ｎ，Ｎ，Ｎ’，Ｎ’－四酢酸（ＥＧＴＡ）、１，２－ビス（ｏ－アミノフェノキシ）エタン－Ｎ，Ｎ，Ｎ’，Ｎ’－四酢酸（ＢＡＰＴＡ）のようなキレート剤を含んでいてもよい。
Furthermore, it can also be used in combination with substances known to promote RT-PCR in the art. Accelerators useful in the present invention include, for example, glycerol, polyols, protease inhibitors, single strand binding proteins (SSBs), T4 gene 32 proteins, tRNA, sulfur or acetic acid-containing compounds, dimethylsulfoxide (DMSO), glycerol, ethylene. Glycerol, Propylene Glycol, Trimethylene Glycol, Formamide, Acetamide, Betaine, Ectoin, Trehalose, Dextran, Polyvinylpyrrolidone (PVP), Tetramethylammonium Chloride (TMC), Tetramethylammonium Hydroxide (TMAH), Tetramethylammonium Acetate (TMAA) ), Polyethylene glycol, Triton X-100, Triton X-114, Tween 20, Nonidet P40, Briji58 and the like, but are not limited thereto. Ethyleneglycol-bis (2-aminoethyl ether) -N, N, N', N'-tetraacetic acid (EGTA), 1,2-bis (o-aminophenoxy) ethane-to further reduce reaction inhibition It may contain a chelating agent such as N, N, N', N'-tetraacetic acid (BAPTA).

　本発明の方法において、前記工程（３）において１ステップＲＴ－ＰＣＲ反応液が、ターゲット領域に対応する１以上のプライマー対をさらに含むことが好ましい。本発明に用いられるプライマー対としては、検出対象のＲＮＡウイルスの検出領域（ターゲット領域）に対応するプライマー対であり、一方のプライマーが他方のプライマーのＤＮＡ伸長生成物に互いに相補的である２種一対のプライマーが挙げられる。また、別の態様として、上記プライマーが２対以上含まれる、いわゆるマルチプレックスＰＣＲも挙げられる。さらに、ターゲットとする核酸が亜型からなる場合、縮重プライマーを含んでもよい。本発明でエンベロープＲＮＡウイルスの１種であるコロナウイルス（ＳＡＲＳ－ｎＣＯＶ－２）を検出する場合、プライマー対の例としては、国立感染症研究所が発表している「病原体検出マニュアル２０１９－ｎＣｏＶ」に記載の配列（配列番号１、２、４、５）、アメリカ疾病予防管理センターが発表する「２０１９－Ｎｏｖｅｌ　Ｃｏｒｏｎａｖｉｒｕｓ　（２０１９－ｎＣｏＶ）　Ｒｅａｌ－ｔｉｍｅ　ｒＲＴ－ｐＣＲ　ＰａｎｅｌＰｒｉｍｅｒｓ　ａｎｄ　Ｐｒｏｂｅｓ」（配列番号７、８、１０、１１、１３、１４）が挙げられ、本発明においても好適に使用することができるが、これに限るものではない。前記記載のプライマー配列では、配列番号１および２、配列番号４および５、配列番号７および８、配列番号１０および１１、配列番号１３および１４によりＳＡＲＳ－ｎＣＯＶ－２のヌクレオキャプシドタンパク質（Ｎ）領域を検出する。ＳＡＲＳ－ｎＣＯＶ－２をはじめとするコロナウイルスの検出においては、ヌクレオキャプシド（Ｎ）領域、エンベロープタンパク質（Ｅ）領域、スパイクタンパク質（S）領域、ＲＮＡ－ｄｅｐｅｎｄｅｎｔ ＲＮＡ ｐｏｌｙｍｅｒａｓｅ（ＲｄＲｐ）領域、Ｏｐｅｎ Ｒｅａｄｉｎｇ Ｆｒａｍｅ（ＯＲＦ）領域等の遺伝子を検出の対象とすることができるが、特にこれに限るものではない。使用するプライマーの濃度としては、ＲＴ－ＰＣＲ反応液全体に対して、フォワードプライマーの濃度が０．１μＭ以上３μＭ以下であり、かつ前記リバースプライマーの濃度が０．１μＭ以上３μＭ以下であることが好ましい。より好ましくは、フォワードプライマーの濃度が０．１μＭ以上２μＭ以下であり、かつ前記リバースプライマーの濃度が０．５μＭ以上２μＭ以下である。
In the method of the present invention, it is preferable that the one-step RT-PCR reaction solution further contains one or more primer pairs corresponding to the target region in the step (3). The primer pair used in the present invention is a primer pair corresponding to the detection region (target region) of the RNA virus to be detected, and two types of primers in which one primer is complementary to the DNA extension product of the other primer. A pair of primers can be mentioned. Further, as another embodiment, so-called multiplex PCR in which two or more pairs of the above primers are contained can be mentioned. In addition, if the target nucleic acid consists of subtypes, it may contain degenerate primers. When detecting coronavirus (SARS-nCOV-2), which is one of the enveloped RNA viruses in the present invention, an example of a primer pair is the "Pathogen Detection Manual 2019-nCoV" published by the National Institute of Infectious Diseases. (SEQ ID NOs: 1, 2, 4, 5), "2019-Novel Coronavirus (2019-nCoV) Real-time rRT-pCR PanelPrimers and Probes" (SEQ ID NOs: 7, 8) announced by the American Center for Disease Control and Prevention. 10, 11, 13, 14) can be mentioned and can be suitably used in the present invention, but the present invention is not limited thereto. In the primer sequences described above, the nucleocapsid protein (N) region of SARS-nCOV-2 by SEQ ID NOs: 1 and 2, SEQ ID NOs: 4 and 5, SEQ ID NOs: 7 and 8, SEQ ID NOs: 10 and 11, and SEQ ID NOs: 13 and 14. Is detected. In the detection of coronaviruses such as SARS-nCOV-2, nucleocapsid (N) region, envelope protein (E) region, spike protein (S) region, RNA-dependent RNA polymerase (RdRp) region, Open Reading Frame. Genes such as the (ORF) region can be detected, but the detection is not limited thereto. As the concentration of the primer to be used, it is preferable that the concentration of the forward primer is 0.1 μM or more and 3 μM or less and the concentration of the reverse primer is 0.1 μM or more and 3 μM or less with respect to the entire RT-PCR reaction solution. .. More preferably, the concentration of the forward primer is 0.1 μM or more and 2 μM or less, and the concentration of the reverse primer is 0.5 μM or more and 2 μM or less.

　本発明は、別の態様としては、さらに、少なくとも１種類の標識されたハイブリダイゼーションプローブまたは２本鎖ＤＮＡ結合蛍光化合物を含む検出方法である。これによって、増幅産物の分析を通常の電気泳動ではなく、蛍光シグナルのモニタリングで監視することができ、解析労力が低減される。さらには、反応容器を開放する必要がなく、コンタミネーションのリスク低減が可能である。ウイルスのサブタイプに対応する、それぞれのハイブリダイゼーションプローブを異なる蛍光色素で標識することによって、ウイルスのサブタイプを識別することも可能である。
Another aspect of the invention is a detection method comprising at least one labeled hybridization probe or double-stranded DNA-bound fluorescent compound. As a result, the analysis of the amplified product can be monitored by monitoring the fluorescent signal instead of the usual electrophoresis, and the analysis labor is reduced. Furthermore, it is not necessary to open the reaction vessel, and the risk of contamination can be reduced. It is also possible to identify the viral subtype by labeling each hybridization probe with a different fluorescent dye, which corresponds to the viral subtype.

　２本鎖ＤＮＡ結合蛍光化合物としては、例えば、ＳＹＢＲ（登録商標）　Ｇｒｅｅｎ　Ｉ，ＳＹＢＲ（登録商標）　Ｇｏｌｄ、ＳＹＴＯ－９、ＳＹＴＰ－１３、ＳＹＴＯ－８２（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ），ＥｖａＧｒｅｅｎ（登録商標；Ｂｉｏｔｉｕｍ）、ＬＣＧｒｅｅｎ（Ｉｄａｈｏ），ＬｉｇｈｔＣｙｃｌｅｒ（登録商標）　４８０　ＲｅｓｏＬｉｇｈｔ（Ｒｏｃｈｅ　Ａｐｐｌｉｅｄ　Ｓｃｉｅｎｃｅ）などが挙げられる。
Examples of the double-stranded DNA-bound fluorescent compound include SYBR (registered trademark) Green I, SYBR (registered trademark) Gold, SYTO-9, SYTP-13, SYTO-82 (Life Technologies), and EvaGreen (registered trademark; Biotium). , LCGreen (Idaho), LightCycler (registered trademark) 480 ResoLight (Roche Applied Science) and the like.

　本発明において用いられるハイブリダイゼーションプローブとしては、例えば、ＴａｑＭａｎ加水分解プローブ（米国特許第５，２１０，０１５号公報、米国特許第５，５３８，８４８号公報、米国特許第５，４８７，９７２号公報、米国特許第５，８０４，３７５号公報）、モレキュラービーコン（米国特許第５，１１８，８０１号公報）、ＦＲＥＴハイブリダイゼーションプローブ（国際公開第９７／４６７０７号パンフレット，国際公開第９７／４６７１２号パンフレット，国際公開第９７／４６７１４号パンフレット）などが挙げられる。エンベロープＲＮＡウイルスの１種であるコロナウイルス（ＳＡＲＳ－ｎＣＯＶ－２）検出用のプローブの塩基配列としては、アメリカ疾病予防管理センターが発表する「２０１９－Ｎｏｖｅｌ　Ｃｏｒｏｎａｖｉｒｕｓ　（２０１９－ｎＣｏＶ）　Ｒｅａｌ－ｔｉｍｅ　ＲＴ－ｐＣＲ　ＰａｎｅｌＰｒｉｍｅｒｓ　ａｎｄ　Ｐｒｏｂｅｓ」（配列番号９、１２、１５）および国立感染症研究所が発表している「病原体検出マニュアル２０１９－ｎＣｏＶ」に記載の配列（配列番号３、６）が挙げられ、本発明においても好適に使用することができるが、これに限るものではない。前記記載のプローブ配列ではＳＡＲＳ－ｎＣＯＶ－２のＮ領域を検出する。さらに、ターゲットとする核酸が亜型からなる場合、縮重配列を含んでもよい。ＳＡＲＳ－ｎＣＯＶ－２をはじめとするコロナウイルスの検出においては、Ｎ領域、Ｅ領域、Ｓ領域、ＲｄＲｐ領域、ＯＲＦ領域等の遺伝子を検出の対象とすることができるが、特にこれに限るものではない。蛍光標識プローブの濃度としては、０．０１μＭ以上１．０μＭ以下であることが好ましい。より好ましくは、０．０１３μＭ以上０．７５μＭ以下であり、更に好ましくは、０．０２μＭ以上０．５μＭ以下である。
Examples of the hybridization probe used in the present invention include TaqMan hydrolysis probe (US Pat. No. 5,210,015, US Pat. No. 5,538,848, US Pat. No. 5,487,972). , US Pat. No. 5,804,375), Molecular Beacon (US Pat. No. 5,118,801), FRET Hybridization Probe (International Publication No. 97/46707, International Publication No. 97/46712) , International Publication No. 97/46714 pamphlet) and the like. As the base sequence of the probe for detecting coronavirus (SARS-nCOV-2), which is one of the enveloped RNA viruses, "2019-Novel Coronavirus (2019-nCoV) Real-time RT-" announced by the American Center for Disease Control and Prevention The sequences (SEQ ID NOs: 3, 6) described in the "pCR PanelPrimers and Probes" (SEQ ID NOs: 9, 12, 15) and the "Pathogen Detection Manual 2019-nCoV" published by the National Institute of Infectious Diseases are mentioned. However, the present invention is not limited to this. The probe sequence described above detects the N region of SARS-nCOV-2. In addition, if the target nucleic acid consists of subtypes, it may contain degenerate sequences. In the detection of coronaviruses such as SARS-nCOV-2, genes such as N region, E region, S region, RdRp region, ORF region can be detected, but it is not particularly limited to this. No. The concentration of the fluorescently labeled probe is preferably 0.01 μM or more and 1.0 μM or less. More preferably, it is 0.013 μM or more and 0.75 μM or less, and even more preferably 0.02 μM or more and 0.5 μM or less.

　本発明の別の一態様は、試料中のウイルスＲＮＡの検査用キットであって、極性有機溶媒およびアニオン性ポリマーを含む前処理液、並びに逆転写酵素およびＤＮＡポリメラーゼ（あるいは逆転写活性を有するＤＮＡポリメラーゼ）、１ステップＲＴ－ＰＣＲ反応液を含むことを特徴とするエンベロープＲＮＡウイルスの検査用キットである。本発明のウイルスの検査用キットは、少なくとも極性有機溶媒、アニオン性ポリマーを含む試薬、逆転写酵素、ＤＮＡポリメラーゼ、および１ステップＲＴ－ＰＣＲ反応液を含む。本発明のウイルスの検査用キットは、極性有機溶媒とアニオン性ポリマーの両方を含有する試薬を含むものであってもよいし、極性有機溶媒とアニオン性ポリマーとを別々の試薬として含むものであってもよい。検査作業をより簡便に行えるという観点から、好ましくは、極性有機溶媒とアニオン性ポリマーの両方を含有する試薬として提供されることが望ましく、本発明の好適なキットでは、この両方を含有する試薬を含む態様で提供される。１ステップＲＴ－ＰＣＲ反応液には、ベタイン様４級アンモニウム塩、ウシ血清アルブミン、グリセロール、グリコールおよびゼラチンのうちの少なくとも１つを含むことが好ましい。検出対象のＲＮＡウイルスの検出領域に対応するプライマー対、さらには検出対象のＲＮＡウイルスの検出領域に対応するハイブリダイゼーションプローブを含むことが好ましい。本発明のキットは、上記のような各種成分を同じ容器に封入したもの又は別々の容器に封入したものを、例えば一つの包装体に梱包し、当該キットの使用方法に関する情報を含む態様で提供することができる。本発明のキットを使用することにより、迅速、簡便に試料中のＲＮＡウイルスの有無について検査することが可能となる。
Another aspect of the invention is a kit for testing viral RNA in a sample, a pretreatment solution containing a polar organic solvent and an anionic polymer, as well as reverse transcriptase and DNA polymerase (or DNA having reverse transcription activity). Polymerase), a kit for testing enveloped RNA virus, which comprises a one-step RT-PCR reaction solution. The virus testing kit of the present invention contains at least a polar organic solvent, a reagent containing an anionic polymer, a reverse transcriptase, a DNA polymerase, and a one-step RT-PCR reaction solution. The virus test kit of the present invention may contain a reagent containing both a polar organic solvent and an anionic polymer, or may contain a polar organic solvent and an anionic polymer as separate reagents. May be. From the viewpoint of facilitating the inspection work, it is preferable that the reagent is provided as a reagent containing both a polar organic solvent and an anionic polymer, and in a suitable kit of the present invention, a reagent containing both of them is preferably provided. Provided in a including manner. The one-step RT-PCR reaction solution preferably contains at least one of betaine-like quaternary ammonium salt, bovine serum albumin, glycerol, glycol and gelatin. It is preferable to include a primer pair corresponding to the detection region of the RNA virus to be detected, and further to include a hybridization probe corresponding to the detection region of the RNA virus to be detected. The kit of the present invention is provided in an embodiment in which various components as described above are enclosed in the same container or enclosed in separate containers, for example, packed in one package and including information on how to use the kit. can do. By using the kit of the present invention, it becomes possible to quickly and easily inspect the presence or absence of RNA virus in a sample.

　以下、実施例をもって、本発明を具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。
Hereinafter, the present invention will be specifically described with reference to Examples. However, the present invention is not limited to the following examples.

試験例１．ＲＮａｓｅによるＲＴ－ＰＣＲ阻害に対するアニオン性ポリマーの効果

（１）反応液の調製

　以下に示される組成の反応液を基本組成とし、１ステップＲＴ－ＰＣＲにて、ＲＮａｓｅＡ存在下における反応液中のコロナウイルスＲＮＡを検出した。検出試薬として、前処理液以外は、ＳＡＲＳ－ＣｏＶ－２　Ｄｅｔｅｃｔｉｏｎ　Ｋｉｔ　－Ｎ１　ｓｅｔ－（東洋紡）を用いた。なお、本試薬の添付品である検出のためのプライマー・プローブは、アメリカ疾病予防管理センター（ＣＤＣ）発行「２０１９－Ｎｏｖｅｌ　Ｃｏｒｏｎａｖｉｒｕｓ　（２０１９－ｎＣｏＶ）　Ｒｅａｌ－ｔｉｍｅ　ＲＴ－ＰＣＲ　Ｐａｎｅｌ　Ｐｒｉｍｅｒｓ　ａｎｄ　Ｐｒｏｂｅｓ」（Ｅｆｆｅｃｔｉｖｅ：　２４　Ｊａｎ　２０２０）　に記載されているＮ１セットであり、プローブは蛍光標識としてＦＡＭ、消光基としてＢＨＱ１（Ｂｌａｃｋ ｈｏｌｅ ｑｕｅｎｃｈｅｒ）を修飾したものを使用した。

　ＲＴ－ＰＣＲ反応液（４３μＬ）

　　反応液：３０μＬ

　　酵素液：５μＬ

　　プライマー・プローブ液：５μＬ

　　ＲＮａｓｅ ｆｒｅｅ ｗａｔｅｒ：３μＬ

（２）鋳型ＲＮＡおよびＲＮａｓｅ　Ａの添加と前処理

　１００％ ジメチルスルホキシド３μＬに、ＲＮＡｓｅ ｆｒｅｅ ｗａｔｅｒまたは１ｎｇ／μＬ　ＲＮａｓｅ　Ａ（ナカライテスク）を１μＬ添加し、これにＲＮＡｓｅ ｆｒｅｅ ｗａｔｅｒまたは、ポリビニルスルホン酸ナトリウム（ＰＶＳＡ）を終濃度０．００１％となるように１μＬ混合した。その後、ＡｃｒｏＭｅｔｒｉｘ　Ｃｏｒｏｎａｖｉｒｕｓ　２０１９（ＣＯＶＩＤ－１９）　ＲＮＡ　Ｃｏｎｔｒｏl（サーモフィッシャーサイエンティフィック）を最終濃度５０コピー／反応～５コピー／反応となるように２μＬを混合し、直ちに混合液７μＬをサーマルサイクラーにて９５℃５分間の熱処理を行った。

（３）反応液の添加

　前工程にて熱処理後の混合液７μＬに、（１）にて調製したＲＴ－ＰＣＲ反応液４３μＬを添加して、５０μＬ反応系にてＲＴ－ＰＣＲを実施した。（４）ＲＴ－ＰＣＲ反応条件　ＳｔｅｐＯｎｅ　ｐｌｕｓ（サーモフィッシャーサイエンティフィック）を使用して、以下の温度サイクルでリアルタイムＰＣＲ反応を実施した。

　４２℃　５分（逆転写条件）

　９５℃　１０秒（熱変性）

　９５℃　１秒－５０℃ ３秒－５５℃　１０秒 ５０サイクル（ＰＣＲ－蛍光読み取り）

（５）結果

　測定結果は、ＳｔｅｐＯｎｅ　ｐｌｕｓ（サーモフィッシャーサイエンティフィック）付属の解析ソフトウェアにて、閾値を１０,０００としてＣｔ値を算出した。この結果を下記の表１及び図１に示す。この結果に示されるように、ＲＮａｓｅ　Ａを添加すると、ＰＶＳＡが存在しない条件では、５０～５コピーすべてにおいて未検出となった。反面、ＰＶＳＡ存在下においては、５０コピー～５コピーが検出可能となった。

Figure JPOXMLDOC01-appb-T000001
Test example 1. Effect of anionic polymers on RT-PCR inhibition by RNase

(1) Preparation of reaction solution

Using the reaction solution having the composition shown below as the basic composition, coronavirus RNA in the reaction solution in the presence of RNase A was detected by 1-step RT-PCR. As the detection reagent, SARS-CoV-2 Detection Kit -N1 set- (Toyobo) was used except for the pretreatment liquid. The primer probe for detection, which is an accessory of this reagent, is "2019-Novell Coronavirus (2019-nCoV) Real-time RT-PCR Panel Primers and Probes" (Effectives) published by the Centers for Disease Control and Prevention (CDC). : 24 Jan 2020), the probe used was FAM as a fluorescent label and BHQ1 (Black hole quencher) modified as a quenching group.

RT-PCR reaction solution (43 μL)

Reaction solution: 30 μL

Enzyme solution: 5 μL

Primer / probe solution: 5 μL

RNase free water: 3 μL

(2) Addition and pretreatment of template RNA and RNase A

Add 1 μL of RNAse free water or 1 ng / μL RNase A (Nacalai Tesque) to 3 μL of 100% dimethyl sulfoxide, and add RNAse free water or sodium polyvinylsulfonate (PVSA) to the final concentration of 0.001%. 1 μL was mixed. Then, 2 μL of AcroMetrix Coronavirus 2019 (COVID-19) RNA Control (Thermo Fisher Scientific) was mixed so as to have a final concentration of 50 copies / reaction to 5 copies / reaction, and 7 μL of the mixed solution was immediately added to 95 in a thermal cycler. Heat treatment was performed at ° C for 5 minutes.

(3) Addition of reaction solution

To 7 μL of the mixed solution after heat treatment in the previous step, 43 μL of the RT-PCR reaction solution prepared in (1) was added, and RT-PCR was carried out in a 50 μL reaction system. (4) RT-PCR reaction conditions Using StepOne plus (Thermo Fisher Scientific), a real-time PCR reaction was carried out in the following temperature cycle.

42 ° C for 5 minutes (reverse transcription conditions)

95 ° C for 10 seconds (heat denaturation)

95 ° C 1 second-50 ° C 3 seconds-55 ° C 10 seconds 50 cycles (PCR-fluorescence reading)

(5) Result

For the measurement results, the Ct value was calculated with the threshold value set to 10,000 by the analysis software attached to StepOne plus (Thermo Fisher Scientific). The results are shown in Table 1 and FIG. 1 below. As shown in this result, the addition of RNase A resulted in undetection in all 50-5 copies in the absence of PVSA. On the other hand, in the presence of PVSA, 50 to 5 copies can be detected.

Figure JPOXMLDOC01-appb-T000001

試験例２．不活化ウイルスを用いたアニオン性ポリマーの効果検討

　以下に示される組成の反応液を基本組成とし、１ステップＲＴ－ＰＣＲにて、ＲＮａｓｅＡ存在下における反応液中の不活化コロナウイルスを検出した。検出試薬として、前処理液以外は、ＳＡＲＳ－ＣｏＶ－２　Ｄｅｔｅｃｔｉｏｎ　Ｋｉｔ　－Ｎ１　ｓｅｔ－（東洋紡）を用いた。なお、本試薬の添付品である検出のためのプライマー・プローブは、アメリカ疾病予防管理センター（ＣＤＣ）発行「２０１９－Ｎｏｖｅｌ　Ｃｏｒｏｎａｖｉｒｕｓ　（２０１９－ｎＣｏＶ）　Ｒｅａｌ－ｔｉｍｅ　ＲＴ－ＰＣＲ　Ｐａｎｅｌ　Ｐｒｉｍｅｒｓ　ａｎｄ　Ｐｒｏｂｅｓ」（Ｅｆｆｅｃｔｉｖｅ：　２４　Ｊａｎ　２０２０）　に記載されているＮ１セットであり、プローブは蛍光標識としてＦＡＭ、消光基としてＢＨＱ１（Ｂｌａｃｋ ｈｏｌｅ ｑｕｅｎｃｈｅｒ）を修飾したものを使用した。

　ＲＴ－ＰＣＲ反応液（４１μＬ）

　　反応液：３０μＬ

　　酵素液：５μＬ

　　プライマー・プローブ液：５μＬ

　　ＲＮａｓｅ ｆｒｅｅ ｗａｔｅｒ：１μＬ

（２）不活化ウイルスおよびＲＮａｓｅ　Ａの添加と前処理

　１００％ ジメチルスルホキシド３μＬに、ＲＮＡｓｅ ｆｒｅｅ ｗａｔｅｒまたは１００ｎｇ／μＬ　ＲＮａｓｅ　Ａ（ナカライテスク）を１μＬ添加し、これにＲＮＡｓｅ ｆｒｅｅ ｗａｔｅｒまたは、ポリビニルスルホン酸ナトリウム（ＰＶＳＡ）を終濃度０．００１％となるように１μＬ混合した。その後、不活化されたコロナウイルス検体として、ＡｃｃｕＰｌｅｘ　ＳＡＲＳ－ＣｏＶ－２　Ｒｅｆｅｒｅｎｃｅ　Ｍａｔｅｒｉａｌ　Ｋｉｔ（Ｓｅｒａｃａｒｅ）添付のＰｏｓｉｔｉｖｅ　ｃｏｎｔｒｏｌを最終濃度２０コピー／反応となるように４μＬを混合し、直ちに混合液９μＬをサーマルサイクラーにて９５℃５分間の熱処理を行った。

（３）反応液の添加

　前工程にて熱処理後の混合液９μＬに、（１）にて調製したＲＴ－ＰＣＲ反応液４１μＬを添加して、５０μＬ反応系にてＲＴ－ＰＣＲを実施した。

（４）ＲＴ－ＰＣＲ反応条件

　ＳｔｅｐＯｎｅ　ｐｌｕｓ（サーモフィッシャーサイエンティフィック）を使用して、以下の温度サイクルでリアルタイムＰＣＲ反応を実施した。

　４２℃　５分（逆転写条件）

　９５℃　１０秒（熱変性）

　９５℃　１秒－５０℃ ３秒－５５℃　１０秒 ５０サイクル（ＰＣＲ－蛍光読み取り）

（５）結果

　測定結果は、ＳｔｅｐＯｎｅ　ｐｌｕｓ（サーモフィッシャーサイエンティフィック）付属の解析ソフトウェアにて、閾値を１０,０００としてＣｔ値を算出した。この結果を下記の表２及び図２に示す。この結果に示されるように、ＲＮａｓｅ　Ａを添加すると、ＰＶＳＡが存在しない条件では、不活化ウイルス２０コピー（n＝４）すべてにおいて未検出となった。反面、ＰＶＳＡ存在下においてはすべて検出可能となった。

Figure JPOXMLDOC01-appb-T000002
Test example 2. Examination of the effect of anionic polymer using inactivated virus

Using the reaction solution having the composition shown below as the basic composition, inactivated coronavirus in the reaction solution in the presence of RNase A was detected by 1-step RT-PCR. As the detection reagent, SARS-CoV-2 Detection Kit -N1 set- (Toyobo) was used except for the pretreatment liquid. The primer probe for detection, which is an accessory of this reagent, is "2019-Novell Coronavirus (2019-nCoV) Real-time RT-PCR Panel Primers and Probes" (Effectives) published by the Centers for Disease Control and Prevention (CDC). : 24 Jan 2020), the probe used was FAM as a fluorescent label and BHQ1 (Black hole quencher) modified as a quenching group.

RT-PCR reaction solution (41 μL)

Reaction solution: 30 μL

Enzyme solution: 5 μL

Primer / probe solution: 5 μL

RNase free water: 1 μL

(2) Addition and pretreatment of inactivated virus and RNase A

Add 1 μL of RNAse free water or 100 ng / μL RNase A (Nacalai Tesque) to 3 μL of 100% dimethyl sulfoxide, and add RNAse free water or sodium polyvinylsulfonate (PVSA) to the final concentration of 0.001%. 1 μL was mixed. Then, as an inactivated coronavirus sample, 4 μL of Positive control attached to AccuPlex SARS-CoV-2 Reference Material Kit (Saracare) was mixed so that the final concentration was 20 copies / reaction, and 9 μL of the mixed solution was immediately added to the thermal cycler. Heat treatment was performed at 95 ° C. for 5 minutes.

(3) Addition of reaction solution

To 9 μL of the mixed solution after heat treatment in the previous step, 41 μL of the RT-PCR reaction solution prepared in (1) was added, and RT-PCR was carried out in a 50 μL reaction system.

(4) RT-PCR reaction conditions

A real-time PCR reaction was performed using StepOne plus (Thermo Fisher Scientific) in the following temperature cycles.

42 ° C for 5 minutes (reverse transcription conditions)

95 ° C for 10 seconds (heat denaturation)

95 ° C 1 second-50 ° C 3 seconds-55 ° C 10 seconds 50 cycles (PCR-fluorescence reading)

(5) Result

For the measurement results, the Ct value was calculated with the threshold value set to 10,000 by the analysis software attached to StepOne plus (Thermo Fisher Scientific). The results are shown in Table 2 and FIG. 2 below. As shown in this result, the addition of RNase A resulted in undetection in all 20 copies of inactivated virus (n = 4) in the absence of PVSA. On the other hand, it became detectable in the presence of PVSA.

Figure JPOXMLDOC01-appb-T000002

試験例３．唾液検体を用いた検討

　以下に示される組成の反応液を基本組成とし、１ステップＲＴ－ＰＣＲにて、唾液検体存在下における反応液中の不活化コロナウイルスを検出した。検出試薬として、前処理液以外は、ＳＡＲＳ－ＣｏＶ－２　Ｄｅｔｅｃｔｉｏｎ　Ｋｉｔ　－Ｎ１　ｓｅｔ－（東洋紡）を用いた。なお、本試薬の添付品である検出のためのプライマー・プローブは、アメリカ疾病予防管理センター（ＣＤＣ）発行「２０１９－Ｎｏｖｅｌ　Ｃｏｒｏｎａｖｉｒｕｓ　（２０１９－ｎＣｏＶ）Ｒｅａｌ－ｔｉｍｅ　ＲＴ－ＰＣＲ　Ｐａｎｅｌ　Ｐｒｉｍｅｒｓ　ａｎｄ　Ｐｒｏｂｅｓ」（Ｅｆｆｅｃｔｉｖｅ：　２４　Ｊａｎ　２０２０）に記載されているＮ１セットであり、プローブは蛍光標識としてＦＡＭ、消光基としてＢＨＱ１（Ｂｌａｃｋ ｈｏｌｅ ｑｕｅｎｃｈｅｒ）を修飾したものを使用した。

　ＲＴ－ＰＣＲ反応液（４１μＬ）

　　反応液：３０μＬ

　　酵素液：５μＬ

　　プライマー・プローブ液：５μＬ

　　ＲＮａｓｅ ｆｒｅｅ ｗａｔｅｒ：１μＬ

（２）唾液検体の添加と不活化ウイルスの前処理

　１００％ ジメチルスルホキシド３μＬに、ＲＮＡｓｅ ｆｒｅｅ ｗａｔｅｒまたは唾液１μＬ添加し、これにＲＮＡｓｅ ｆｒｅｅ ｗａｔｅｒまたは、ポリビニルスルホン酸ナトリウム（ＰＶＳＡ）を終濃度０．００１％となるように１μＬ混合した。その後、不活化されたコロナウイルス検体として、ＡｃｃｕＰｌｅｘ　ＳＡＲＳ－ＣｏＶ－２　Ｒｅｆｅｒｅｎｃｅ　Ｍａｔｅｒｉａｌ　Ｋｉｔ（Ｓｅｒａｃａｒｅ）添付のＰｏｓｉｔｉｖｅ　ｃｏｎｔｒｏｌを最終濃度２０コピー／反応となるように４μＬを混合し、直ちに混合液９μＬをサーマルサイクラーにて９５℃５分間の熱処理を行った。

（３）反応液の添加

　前工程にて熱処理後の混合液９μＬに、（１）にて調製したＲＴ－ＰＣＲ反応液４１μＬを添加して、５０μＬ反応系にてＲＴ－ＰＣＲを実施した。

（４）ＲＴ－ＰＣＲ反応条件

　ＳｔｅｐＯｎｅ　ｐｌｕｓ（サーモフィッシャーサイエンティフィック）を使用して、以下の温度サイクルでリアルタイムＰＣＲ反応を実施した。

　４２℃　５分（逆転写条件）

　９５℃　１０秒（熱変性）

　９５℃　１秒－５０℃ ３秒－５５℃　１０秒 ５０サイクル（ＰＣＲ－蛍光読み取り）

（５）結果

　測定結果は、ＳｔｅｐＯｎｅ　ｐｌｕｓ（サーモフィッシャーサイエンティフィック）付属の解析ソフトウェアにて、閾値を１０,０００としてＣｔ値を算出した。この結果を下記の表３及び図３に示す。この結果に示されるように、ＲＮａｓｅ　Ａを添加すると、ＰＶＳＡが存在しない条件では、不活化ウイルス２０コピー（ｎ＝４）すべてにおいて未検出となった。反面、ＰＶＳＡ存在下においてはｎ＝４中で３サンプルにおいて検出が確認された。

Figure JPOXMLDOC01-appb-T000003
Test example 3. Examination using saliva samples

Using the reaction solution having the composition shown below as the basic composition, inactivated coronavirus in the reaction solution in the presence of a saliva sample was detected by 1-step RT-PCR. As the detection reagent, SARS-CoV-2 Detection Kit -N1 set- (Toyobo) was used except for the pretreatment liquid. The primer probe for detection, which is an accessory of this reagent, is "2019-Novel Coronavirus (2019-nCoV) Real-time RT-PCR Panel Primers and Probes" (Effectives) published by the Centers for Disease Control and Prevention (CDC). : 24 Jan 2020), the probe used was FAM as a fluorescent label and BHQ1 (Black hole quencher) modified as a quenching group.

RT-PCR reaction solution (41 μL)

Reaction solution: 30 μL

Enzyme solution: 5 μL

Primer / probe solution: 5 μL

RNase free water: 1 μL

(2) Addition of saliva sample and pretreatment of inactivated virus

To 3 μL of 100% dimethyl sulfoxide, 1 μL of RNAse free water or saliva was added, and 1 μL of RNAse free water or sodium polyvinylsulfonate (PVSA) was mixed with the final concentration of 0.001%. Then, as an inactivated coronavirus sample, 4 μL of Positive control attached to AccuPlex SARS-CoV-2 Reference Material Kit (Saracare) was mixed so that the final concentration was 20 copies / reaction, and 9 μL of the mixed solution was immediately added to the thermal cycler. Heat treatment was performed at 95 ° C. for 5 minutes.

(3) Addition of reaction solution

To 9 μL of the mixed solution after heat treatment in the previous step, 41 μL of the RT-PCR reaction solution prepared in (1) was added, and RT-PCR was carried out in a 50 μL reaction system.

(4) RT-PCR reaction conditions

A real-time PCR reaction was performed using StepOne plus (Thermo Fisher Scientific) in the following temperature cycles.

42 ° C for 5 minutes (reverse transcription conditions)

95 ° C for 10 seconds (heat denaturation)

95 ° C 1 second-50 ° C 3 seconds-55 ° C 10 seconds 50 cycles (PCR-fluorescence reading)

(5) Result

For the measurement results, the Ct value was calculated with the threshold value set to 10,000 by the analysis software attached to StepOne plus (Thermo Fisher Scientific). The results are shown in Table 3 and FIG. 3 below. As shown in this result, the addition of RNase A resulted in undetection in all 20 copies of inactivated virus (n = 4) in the absence of PVSA. On the other hand, in the presence of PVSA, detection was confirmed in 3 samples in n = 4.

Figure JPOXMLDOC01-appb-T000003

　本発明は、分子生物学研究、さらに臨床検査や食品衛生管理などを目的とした検査において、好適に用いられる。
INDUSTRIAL APPLICABILITY The present invention is suitably used in molecular biology research, and in tests for the purpose of clinical tests, food hygiene control, and the like.

Claims (31)


  1. 　試料中のＲＮＡウイルスの検査方法であって、以下の工程を含むことを特徴とする検査方法。

    （１）ＲＮＡの精製を行っていない試料と、アニオン性ポリマーと、極性有機溶媒とを含む混合液を調製する工程、

    （２）前記混合液を加熱する工程、

    （３）加熱後の前記混合液に、（ｉ）逆転写酵素およびＤＮＡポリメラーゼまたは（ｉｉ）逆転写活性を有するＤＮＡポリメラーゼを含む１ステップＲＴ－ＰＣＲ反応液を添加する工程、

    （４）反応容器を密閉後、１ステップＲＴ－ＰＣＲ反応を実施する工程。
    A method for inspecting RNA virus in a sample, which comprises the following steps.

    (1) A step of preparing a mixed solution containing a sample in which RNA has not been purified, an anionic polymer, and a polar organic solvent.

    (2) Step of heating the mixed solution,

    (3) A step of adding a one-step RT-PCR reaction solution containing (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcription activity to the heated mixed solution.

    (4) A step of carrying out a one-step RT-PCR reaction after sealing the reaction vessel.

  2. 　工程（１）において、混合液中の極性有機溶媒の含有率が２０％以上である請求項１に記載の検査方法。
    The inspection method according to claim 1, wherein in the step (1), the content of the polar organic solvent in the mixed solution is 20% or more.

  3. 　工程（１）において、混合液中のアニオン性ポリマーの含有率が０．００００１％以上である請求項１又は２に記載の検査方法。
    The inspection method according to claim 1 or 2, wherein in the step (1), the content of the anionic polymer in the mixed solution is 0.00001% or more.

  4. 　工程（１）において、混合液が界面活性剤を実質的に含まないことを特徴とする請求項１から請求項３のいずれかに記載の検査方法。
    The inspection method according to any one of claims 1 to 3, wherein in the step (1), the mixed solution contains substantially no surfactant.

  5. 　工程（１）において混合液を調製後、工程（２）を実施するまでの時間が５分以内である請求項１から４のいずれかに記載の検査方法。
    The inspection method according to any one of claims 1 to 4, wherein the time from preparing the mixed solution in the step (1) to carrying out the step (2) is within 5 minutes.

  6. 　工程（２）における加熱条件が、７０℃１秒以上であることを特徴とする請求項１から５のいずれかに記載の検査方法。
    The inspection method according to any one of claims 1 to 5, wherein the heating condition in the step (2) is 70 ° C. for 1 second or more.

  7. 　試料が糞便、咽頭ぬぐい液、鼻腔ぬぐい液、喀痰、肺吸引物、脳脊髄液、うがい液、唾液、涙液、培養細胞、培養上清、及び環境中の拭き取り検査試料からなる群より選択される少なくとも１種である請求項１から６のいずれかに記載の検査方法。
    Samples are selected from the group consisting of feces, pharyngeal swabs, nasal swabs, sputum, lung aspirates, cerebrospinal fluid, mouthwash, saliva, tears, cultured cells, culture supernatants, and environmental wiping test samples. The inspection method according to any one of claims 1 to 6, which is at least one kind.

  8. 　試料が水、生理食塩水、緩衝液若しくはスプタザイム酵素液に懸濁された懸濁液、又はそれらの遠心上清若しくは濃縮物である請求項１から７のいずれかに記載の検査方法。
    The test method according to any one of claims 1 to 7, wherein the sample is a suspension suspended in water, physiological saline, a buffer solution or a sputazyme enzyme solution, or a centrifugal supernatant or a concentrate thereof.

  9. 　ＲＮＡウイルスがエンベロープを持つＲＮＡウイルスであることを特徴とする請求項１から請求項８のいずれかに記載の検査方法。
    The test method according to any one of claims 1 to 8, wherein the RNA virus is an RNA virus having an envelope.

  10. 　エンベロープを持つＲＮＡウイルスが、フラビウイルス科ウイルス；トガウイルス科ウイルス；コロナウイルス科ウイルス；オルトミクソウイルス科ウイルス；ラブドウイルス科ウイルス；ブニヤウイルス科ウイルス；パラミクソウイルス科ウイルス；及びフィロウイルス科ウイルスからなる群より選択されることを特徴とする請求項９に記載の検査方法。
    The enveloped RNA virus consists of flavivirus family virus; togavirus family virus; coronavirus family virus; orthomixovirus family virus; rabdovirus family virus; bunyavirus family virus; paramyxovirus family virus; and phyllovirus family virus. The inspection method according to claim 9, wherein the virus is selected from the group.

  11. 　エンベロープを持つＲＮＡウイルスがコロナウイルス科ウイルスである請求項１から１０のいずれかに記載の検査方法。
    The test method according to any one of claims 1 to 10, wherein the RNA virus having an envelope is a coronaviridae virus.

  12. 　コロナウイルス科ウイルスがＳＡＲＳ（重症急性呼吸器症候群）コロナウイルス、ＭＥＲＳ（中東呼吸器症候群）コロナウイルス、ＳＡＲＳ－ｎＣＯＶ－２コロナウイルスである請求項１１に記載の検査方法。
    The test method according to claim 11, wherein the coronavirus family virus is SARS (severe acute respiratory syndrome) coronavirus, MERS (Middle East respiratory syndrome) coronavirus, and SARS-nCOV-2 coronavirus.

  13. 　ＲＮＡウイルスがエンベロープを持たないＲＮＡウイルスであることを特徴とする請求項１から請求項１２のいずれかに記載の検査方法。
    The test method according to any one of claims 1 to 12, wherein the RNA virus is an RNA virus having no envelope.

  14. 　エンベロープを持たないＲＮＡウイルスが、アストロウイルス科ウイルス；カリシウイルス科ウイルス；ピコルナウイルス科ウイルス；へペウイルス科ウイルス；及びレオウイルス科ウイルスからなる群より選択されることを特徴とする請求項１３に記載のウイルスの検査方法。
    13. Claim 13, wherein the RNA virus having no envelope is selected from the group consisting of astroviridae virus; caliciviridae virus; picornaviridae virus; hepeviridae virus; and leoviridae virus. The virus inspection method described.

  15. 　極性有機溶媒が、エタノール、メタノール、１－プロパノール、２－プロパノール、１－ブタノール、２－ブタノール、ピリジン、トリエチルアミンジメチルホルムアミド、ヘキサメチルホスホリックトリアミド、ジメチルスルホキシド、アセトン、およびアセトニトリルよりなる群から選択される少なくとも１種であることを特徴とする請求項１から１４のいずれかに記載の検査方法。
    The polar organic solvent is selected from the group consisting of ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pyridine, triethylamine dimethylformamide, hexamethylphosphoric triamide, dimethyl sulfoxide, acetone, and acetonitrile. The inspection method according to any one of claims 1 to 14, wherein the inspection method is at least one thereof.

  16. 　アニオン性ポリマーが、スルホン酸基、カルボキシル基、リン酸基、硫酸基、及びホスホン酸基からなる群より選択される少なくとも１種のアニオン性官能基を有するモノマーを重合して得られるポリマーである請求項１から請求項１５のいずれかに記載の検査方法。
    The anionic polymer is a polymer obtained by polymerizing a monomer having at least one anionic functional group selected from the group consisting of a sulfonic acid group, a carboxyl group, a phosphoric acid group, a sulfuric acid group, and a phosphonic acid group. The inspection method according to any one of claims 1 to 15.

  17. 　アニオン性ポリマーが、ポリイノシン酸、ポリシチジル酸、ポリグアニル酸、ポリアデニル酸、ポリデオキシイノシン酸、ポリデオキシシチジル酸、ポリデオキシグアニル酸、ポリデオキシアデニル酸、カラギーナン、ヘパリン、コンドロイチン硫酸、ケラタン硫酸、ヒアルロン酸、ヘパラン硫酸、コンドロイチン、デルマタン硫酸、ポリビニルスルホン酸、ポリビニルホスホン酸、ポリスチレンスルホン酸、ポリアクリル酸、ポリアクリル酸／スルホン酸共重合体、ポリアクリル酸／マレイン酸共重合体およびこれらの塩からなる群から選択される少なくとも１種のアニオン性ポリマーである請求項１から１６のいずれかに記載のウイルスの検査方法。
    Anionic polymers include polyinosic acid, polycitidilic acid, polyguanylic acid, polyadenylic acid, polydeoxyinosic acid, polydeoxycitidilic acid, polydeoxyguanyl acid, polydeoxyadenylic acid, carrageenan, heparin, chondroitin sulfate, keratane sulfate, hyaluronic acid. , Heparan sulfate, chondroitin, dermatane sulfate, polyvinyl sulfonic acid, polyvinyl phosphonic acid, polystyrene sulfonic acid, polyacrylic acid, polyacrylic acid / sulfonic acid copolymer, polyacrylic acid / maleic acid copolymer and salts thereof. The method for testing a virus according to any one of claims 1 to 16, which is at least one anionic polymer selected from the group.

  18. 　ＤＮＡポリメラーゼが、Ｔａｑ、Ｔｔｈおよびそれらの変異体よりなる群から選択されるいずれかであることを特徴とする請求項１から１７のいずれかに記載の検査方法。
    The test method according to any one of claims 1 to 17, wherein the DNA polymerase is any one selected from the group consisting of Taq, Tth and variants thereof.

  19. 　逆転写酵素の由来が、モロニーマウス白血病ウイルス（ＭＭＲＶ）、トリ骨髄芽球症ウイルス（ＡＭＶ）およびこれらの変異体からなる群より選択されるいずれかであることを特徴とする請求項１から１８のいずれかに記載の検査方法。
    Claims 1-18, wherein the reverse transcriptase is derived from any one selected from the group consisting of Moloney murine leukemia virus (MMRV), avian myeloblastosis virus (AMV) and variants thereof. The inspection method described in any of.

  20. 　工程（４）における１ステップＲＴ－ＰＣＲ反応液が、アミノ酸におけるアミノ基に３個のメチル基を付加した構造を有する第４級アンモニウム塩（以下、「ベタイン様４級アンモニウム」という）、ウシ血清アルブミン、グリセロール、グリコールおよびゼラチンよりなる群から選択された少なくとも１つをさらに含むことを特徴とする請求項１から１９のいずれかに記載の検査方法。
    The 1-step RT-PCR reaction solution in step (4) is a quaternary ammonium salt having a structure in which three methyl groups are added to an amino group in an amino acid (hereinafter referred to as "betaine-like quaternary ammonium"), bovine serum. The test method according to any one of claims 1 to 19, further comprising at least one selected from the group consisting of albumin, glycerol, glycol and gelatin.

  21. 　ベタイン様４級アンモニウム塩が、ベタインまたはＬ－カルニチンである請求項２０に記載のウイルスの検査方法。
    The method for testing a virus according to claim 20, wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine.

  22. 　アニオン性ポリマー、極性有機溶媒、（ｉ）逆転写酵素及びＤＮＡポリメラーゼ又は（ｉｉ）逆転写酵素活性を有するＤＮＡポリメラーゼを含む１ステップＲＴ－ＰＣＲ反応液を含むことを特徴とするＲＮＡウイルスの検査用キット。
    For testing RNA viruses comprising a one-step RT-PCR reaction solution comprising an anionic polymer, a polar organic solvent, (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcriptase activity. kit.

  23. 　ベタイン様４級アンモニウム塩、ウシ血清アルブミン、グリセロール、グリコールおよびゼラチンよりなる群から選択された少なくとも１つをさらに含む請求項２２に記載の検査用キット。
    22. The test kit according to claim 22, further comprising at least one selected from the group consisting of betaine-like quaternary ammonium salts, bovine serum albumin, glycerol, glycols and gelatin.

  24. 　検出対象のＲＮＡウイルスの検出領域に対応するプライマー対をさらに含むことを特徴とする請求項２２または２３に記載のウイルスの検査用キット。
    The virus testing kit according to claim 22 or 23, further comprising a primer pair corresponding to the detection region of the RNA virus to be detected.

  25. 　検出対象のＲＮＡウイルスの検出領域に対応するハイブリダイゼーションプローブをさらに含むことを特徴とする請求項２２から２４いずれかに記載のウイルスの検査用キット。
    The virus testing kit according to any one of claims 22 to 24, further comprising a hybridization probe corresponding to the detection region of the RNA virus to be detected.

  26. 　ＲＮＡウイルスがエンベロープを持つことを特徴とする請求項２２から２５のいずれかに記載のウイルスの検査キット。
    The virus test kit according to any one of claims 22 to 25, wherein the RNA virus has an envelope.

  27. 　エンベロープを持つＲＮＡウイルスが、フラビウイルス科ウイルス；トガウイルス科ウイルス；コロナウイルス科ウイルス；オルトミクソウイルス科ウイルス；ラブドウイルス科ウイルス；ブニヤウイルス科ウイルス；パラミクソウイルス科ウイルス；及びフィロウイルス科ウイルスからなる群より選択されることを特徴とする請求項２６に記載のウイルスの検査用キット。
    The enveloped RNA virus consists of flavivirus family virus; togavirus family virus; coronavirus family virus; orthomixovirus family virus; rabdovirus family virus; bunyavirus family virus; paramyxovirus family virus; and phyllovirus family virus. The virus testing kit according to claim 26, which is selected from the group.

  28. 　エンベロープを持つＲＮＡウイルスがコロナウイルスである請求項２６又は２７のいずれかに記載のウイルスの検査用キット。
    The virus testing kit according to claim 26 or 27, wherein the RNA virus having an envelope is a coronavirus.

  29. 　コロナウイルス科ウイルスがＳＡＲＳ（重症急性呼吸器症候群）コロナウイルス、ＭＥＲＳ（中東呼吸器症候群）コロナウイルス、ＳＡＲＳ－ｎＣＯＶ－２であることを特徴とする請求項２７又は請求項２８に記載のウイルスの検査用キット。
    The virus according to claim 27 or 28, wherein the coronavirus family virus is SARS (severe acute respiratory syndrome) coronavirus, MERS (Middle East respiratory syndrome) coronavirus, SARS-nCOV-2. Inspection kit.

  30. 　ＲＮＡウイルスがエンベロープを持たないことを特徴とする請求項２２から２５のいずれかに記載のウイルスの検査キット。
    The virus test kit according to any one of claims 22 to 25, wherein the RNA virus has no envelope.

  31. 　エンベロープを持たないＲＮＡウイルスが、アストロウイルス科ウイルス；カリシウイルス科ウイルス；ピコルナウイルス科ウイルス；へペウイルス科ウイルス；及びレオウイルス科ウイルスからなる群より選択されることを特徴とする請求項３０に記載のウイルスの検査用キット。
    30 is characterized in that the non-enveloped RNA virus is selected from the group consisting of astroviridae virus; caliciviridae virus; picornaviridae virus; hepeviridae virus; and leoviridae virus. The virus test kit described.
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