WO2022024934A1 - 改良されたウイルスの検出方法 - Google Patents

改良されたウイルスの検出方法 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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French (fr)
Japanese (ja)
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奈々 山越
旦 岡山
謙太 寺内
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Toyobo Co Ltd
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Toyobo Co Ltd
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Priority to CN202180059316.6A priority patent/CN116529362A/zh
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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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