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WO2021200717A1 - Improved method of virus detection - Google Patents

Improved method of virus detection Download PDF

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Publication number
WO2021200717A1
WO2021200717A1 PCT/JP2021/013045 JP2021013045W WO2021200717A1 WO 2021200717 A1 WO2021200717 A1 WO 2021200717A1 JP 2021013045 W JP2021013045 W JP 2021013045W WO 2021200717 A1 WO2021200717 A1 WO 2021200717A1
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Prior art keywords
solution
virus
sample
aqueous solution
coronavirus
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PCT/JP2021/013045
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French (fr)
Japanese (ja)
Inventor
謙太 寺内
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Toyobo Co Ltd
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Toyobo Co Ltd
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Priority to JP2022512146A priority Critical patent/JPWO2021200717A1/ja
Publication of WO2021200717A1 publication Critical patent/WO2021200717A1/en
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • C12Q1/6813—Hybridisation assays
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • 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 a virus by nucleic acid amplification. More specifically, RNA having an envelope is obtained by mixing the sample with an alkaline solution and then adding a reaction solution for real-time reverse transcription-polymerase chain reaction (qRT-PCR) without isolating and purifying the nucleic acid from the sample. Regarding virus detection. More specifically, it relates to a method for detecting a coronavirus. According to the present invention, it is possible to detect coronavirus contained in, for example, pharyngeal swab, nasal swab, biological sample such as sputum, fecal sample, blood sample, environmental wiping sample, etc. with high sensitivity. .. The present invention can be used for life science research, clinical diagnosis, food hygiene inspection, environmental inspection, and the like.
  • 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 Severe Acute Respiratory Syndrome (COVID-19) coronavirus (SARS). It is known that those causing serious fatal 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
  • 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).
  • detection of coronavirus contained in a sample involves extraction and purification of viral RNA from the sample. The process of extracting and purifying viral RNA was complicated and required a lot of working time.
  • RT-PCR reaction inhibitors vary widely depending on the type of sample. For example, PCR reaction inhibitors such as polysaccharides are introduced into stool samples. In addition, it is known that virus inactivation and RNA extraction conditions differ greatly depending on the virus species.
  • RNA separation and purification steps are required in the method of testing by RT-PCR reaction from biological samples such as pharyngeal / nasal swabs and fecal samples containing coronavirus, especially SARS-nCOV-2, and wiping environment samples.
  • biological samples such as pharyngeal / nasal swabs and fecal samples containing coronavirus, especially SARS-nCOV-2, and wiping environment samples.
  • RNA of enveloped viruses especially coronavirus (especially SARS-nCOV-2)
  • coronavirus especially SARS-nCOV-2
  • the present inventors have conducted diligent research and found that a sample in which nucleic acid has not been separated and purified in advance is mixed with an alkaline solution and then subjected to 1-step RT-PCR to be contained in the sample.
  • a virus having an envelope particularly a coronavirus (among others, SARS-nCOV-2), can be detected with sufficient sensitivity, and arrived at the present invention.
  • Item 1 A method for testing an RNA virus having an envelope in a sample, which comprises the following steps: (1) A step of mixing a sample that has not been separated and purified from nucleic acid with an alkaline solution having a pH of 8 or higher. (2) 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 after 1 minute or more has passed from the preparation of the mixed solution. (3) A step of carrying out a one-step RT-PCR reaction after sealing the reaction vessel.
  • Item 2. The inspection method according to Item 1, wherein the steps (1) to (3) are performed in the same container.
  • the alkaline solution is potassium hydroxide aqueous solution, sodium hydroxide aqueous solution, lithium hydroxide aqueous solution, magnesium hydroxide aqueous solution, calcium hydroxide aqueous solution, barium hydroxide aqueous solution, potassium carbonate aqueous solution, sodium carbonate aqueous solution, magnesium carbonate aqueous solution, carbon dioxide.
  • Item 5 At least one sample selected from the group consisting of feces, pharyngeal swab, nasal swab, sputum, pulmonary aspirate, cerebrospinal fluid, mouthwash, saliva, tears, cultured cells, and culture supernatant.
  • Item 7 The inspection method according to Item 6, wherein the sample is a centrifugal supernatant of a suspension.
  • Item 8 The inspection method according to any one of Items 1 to 7, wherein the sample is a sample in which an environmental wiping test sample is previously suspended in water, physiological saline or a buffer solution, and the suspension is concentrated. ..
  • Item 9. The test method according to any one of Items 1 to 8, wherein the RNA virus having an envelope is a coronavirus.
  • Item 10 The test method according to Item 9, wherein the coronavirus is SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, or SARS-nCOV-2 coronavirus.
  • SARS severe Acute Respiratory Syndrome
  • MERS Middle East Respiratory Syndrome
  • the test method according to any one of Items 1 to 10, wherein the DNA polymerase is at least one selected from the group consisting of Taq, Tth and variants thereof.
  • the reverse transcriptase is at least one selected from the group consisting of Moloney murine leukemia virus (MMRV) -derived reverse transcriptase, avian myeloblastosis virus (AMV) -derived reverse transcriptase, and variants thereof.
  • MMRV Moloney murine leukemia virus
  • AMV avian myeloblastosis virus
  • the 1-step RT-PCR reaction solution in step (3) has a structure in which three methyl groups are added to an amino group in an amino acid, and is a quaternary ammonium salt (hereinafter referred to as “betaine-like quaternary ammonium”).
  • the test method according to any one of Items 1 to 12, which comprises at least one selected from the group consisting of bovine serum albumin, glycerol, glycol and gelatin.
  • Item 14 The inspection method according to Item 13, wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine.
  • a kit for testing a virus having an envelope which comprises an alkaline solution having a pH of 8 or higher, a reverse transcriptase, a DNA polymerase, and a 1-step RT-PCR reaction solution.
  • Item 16 The virus inspection kit according to Item 15, which is used for inspecting the presence or absence of an enveloped virus from a sample that has not been subjected to the separation and purification treatment of nucleic acid.
  • Item 17. After 1 minute or more has passed from the preparation of the mixed solution of the sample not subjected to the separation and purification treatment of the nucleic acid and the alkaline solution having a pH of 8 or more, the 1-step RT-PCR reaction solution is added, the reaction vessel is sealed, and then the reaction vessel is sealed.
  • the virus testing kit according to Item 16 which is used to carry out a one-step RT-PCR reaction.
  • the alkaline solution is potassium hydroxide aqueous solution, sodium hydroxide aqueous solution, lithium hydroxide aqueous solution, magnesium hydroxide aqueous solution, calcium hydroxide aqueous solution, barium hydroxide aqueous solution, potassium carbonate aqueous solution, sodium carbonate aqueous solution, magnesium carbonate aqueous solution, calcium carbonate.
  • Items 15 to 17 which is an aqueous solution, a Tris buffer solution, a glycine buffer solution, a phosphate buffer solution, a borate buffer solution, or a Good buffer solution.
  • Item 19 The one-step RT-PCR reaction solution according to any one of Items 15 to 18, which comprises at least one selected from the group consisting of betaine-like quaternary ammonium salt, bovine serum albumin, glycerol, glycol and gelatin.
  • Virus test kit. Item 20. The virus testing kit according to any one of Items 15 to 19, further comprising a primer pair corresponding to the detection region of the RNA virus to be detected.
  • Item 21. The virus testing kit according to any one of Items 15 to 20, further comprising a hybridization probe corresponding to the detection region of the RNA virus to be detected.
  • Item 22 The virus test kit according to any one of Items 15 to 21, wherein the RNA virus having an envelope is a coronavirus.
  • coronavirus is SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, or SARS-nCOV-2. ..
  • the sample is mixed with an alkaline solution and then added to the 1-step RT-PCR reaction solution to obtain an enveloped virus such as coronavirus in the sample.
  • the presence or absence can be easily detected in a short time.
  • the efficiency of inspection work can be further improved, so that the number of inspections can be increased, which contributes to early detection of infectious diseases and prevention of spread of infection.
  • the work of opening and closing the lid of the reaction vessel is also omitted.
  • the risk of scattering of the virus-containing sample when opening and closing the lid can be eliminated, and the risk of contamination to other samples can also be reduced.
  • the risk of false positives can be suppressed, and the accuracy of inspection work can be further improved.
  • the risk of infection to workers can be reduced by eliminating the risk of scattering the virus-containing sample.
  • the present invention exerts an excellent effect in the inspection of a sample that may contain SARS-nCOV-2 (which may also be referred to as SARS-CoV-2, 2019-nCoV, etc.) that occurred in 2019.
  • SARS-nCOV-2 which may also be referred to as SARS-CoV-2, 2019-nCoV, etc.
  • blood, feces excretion, rectal stool
  • vomitus urine
  • sputum lymph
  • lymph plasma
  • ejaculation pulmonary aspirate
  • cerebrospinal fluid pharyngeal swab
  • nasal swab gargling.
  • coronavirus particularly SARS-nCOV-2
  • contaminant-rich samples such as biological samples containing fluids, saliva, tears, environmental wipes, cultured cells or samples containing culture supernatants. Can also be detected.
  • 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 a method for testing an RNA virus having an envelope such as coronavirus (particularly SARS-nCOV-2) in a sample (hereinafter, this is also referred to as "emberobe RNA virus” or the like). Then, without performing separation and purification of viral RNA from the sample in advance, the sample and a reagent containing an alkaline solution are mixed to obtain (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcription activity.
  • the method for inspecting the presence or absence of an enveloped RNA virus in a sample of the present invention is an inspection method comprising at least the following steps.
  • (1) A step of mixing a sample that has not been separated and purified from nucleic acid with a reagent containing an alkaline solution.
  • (2) After 1 minute or more has passed from the preparation of the mixed solution, a 1-step RT-PCR reaction solution containing (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcription activity is added to the mixed solution. Addition process, (3) A step of carrying out a one-step RT-PCR reaction after sealing the reaction vessel. It is preferable that all of the steps (1) and (2), preferably steps (1) to (3), are performed in the same container.
  • the mixed solution it is preferable not to transfer all or a part of the mixed solution to another container between the steps (1) and (2), preferably between the steps (1) to (3).
  • the entire amount of the mixed solution may be subjected to the step (2) according to the step (1), or a part thereof may be transferred to another container to carry out the step (2).
  • One of the features of the present invention is that the mixed solution obtained in the step (1) is reacted in a mixed state for 1 minute or longer.
  • a PCR reaction inhibitor such as a contaminant is contained in the collected biological sample. Even if this is the case, it is possible to detect a virus having an envelope (for example, coronavirus, especially SARS-nCOV-2) that can be contained in the sample in the one-step RT-PCR reaction.
  • the reaction time after preparing the mixed solution is not particularly limited as long as it is 1 minute or more as long as the effects of the present invention are exhibited.
  • the addition of the 1-step RT-PCR reaction solution may be carried out after a lapse of about 1 to 20 minutes in the step (2), or the 1-step RT-PCR reaction may be carried out after a lapse of 20 minutes or more. ..
  • the time from obtaining the mixed solution in step (1) to adding the RT-PCR reaction solution in step (2) is 1 minute or more. It is preferably 10 minutes or less, more preferably 1 minute or more and 5 minutes or less, further preferably 1 minute or more and 4 minutes or less, and particularly preferably 1 minute or more and 3 minutes or less.
  • the state of the mixed solution until the RT-PCR reaction solution of the step (2) is added may be a stationary state or a stirring state using a vortex mixer, a dispenser or the like.
  • the temperature conditions of the mixed solution obtained in the step (1) until the RT-PCR reaction solution is added in the step (2) are not particularly limited. Temperature range such as low temperature (for example, about -10 ° C to 10 ° C (preferably on ice)), room temperature (for example, about 10 ° C to 40 ° C), high temperature (40 ° C or higher) so that the mixed solution does not freeze. Can be mentioned.
  • the reaction is preferably carried out at a low temperature or room temperature, more preferably under temperature conditions of ice to room temperature, and in consideration of workability, the reaction is preferably carried out at room temperature.
  • the method of mixing the sample or its suspension or solution with the alkaline solution is not particularly limited, and for example, a dispenser or the like is used for the alkaline solution previously dispensed in the RT-PCR reaction vessel. Then, a method of adding and mixing the sample, a method of suspending the sampling tool to which the sample is attached, for example, a swab or a stool collecting device in direct contact with an alkaline solution, and the like can be mentioned.
  • the alkaline solution is not particularly limited as long as it is an alkaline solution.
  • the alkaline solution include potassium hydroxide aqueous solution, sodium hydroxide aqueous solution, lithium hydroxide aqueous solution, magnesium hydroxide aqueous solution, calcium hydroxide aqueous solution, barium hydroxide aqueous solution, potassium carbonate aqueous solution, sodium carbonate aqueous solution, magnesium carbonate aqueous solution, and calcium carbonate.
  • Aqueous solution Tris buffer solution, glycine buffer solution, phosphate buffer solution, borate buffer solution, Good buffer solution (for example, TAPSO, POPSO, HEPSO, EPPS, Tricine, Bine, TAPS, CHES, CAPS).
  • Good buffer solution for example, TAPSO, POPSO, HEPSO, EPPS, Tricine, Bine, TAPS, CHES, CAPS.
  • a preferred alkaline solution is at least one solution selected from the group consisting of Tris buffer, Tricine buffer, and Glycine buffer.
  • a more preferred alkaline solution is Tris buffer or Glycine buffer.
  • the alkaline solution is not particularly limited as long as it is an alkaline solution, and for example, the hydrogen ion index (pH) (25 ° C.) is pH 8.0 or higher, pH 8.5 or higher, pH 9.0 or higher, pH 9.5 or higher, pH 10.
  • the pH may be 10.5 or higher, pH 11.0 or higher, pH 11.5 or higher, pH 12.0 or higher, pH 12.5 or higher, pH 13.0 or higher, pH 13.5 or higher, or pH 14.0 or higher.
  • the alkaline solution preferably has a pH of about 8 to 14, more preferably about 8 to 12, and even more preferably about pH 8 to 10. It is preferable that the pH is about 8 to 9.5.
  • the concentration of the alkaline substance (also referred to as "alkaline concentration") of the alkaline solution used in the present invention is not particularly limited as long as it is adjusted to be within the above pH range.
  • the alkaline concentration is a volume molar concentration (M or mol / L), and the total amount of alkaline substances (for example, potassium hydroxide and / or sodium hydroxide as described above) contained in 1 L of the alkaline solution. ) Can be specified by the molar amount.
  • the alkali concentration can be, for example, 1 mM or more, 1.5 mM or more, 2 mM or more, 4 mM or more, 5 mM or more, 6 mM or more, 8 mM or more, or 10 mM or more, and for example, 50 mM or less, 49 mM or less, 48 mM or less, 47 mM or less, It may be 46 mM or less, 45 mM or less, 44 mM or less, 43 mM or less, 42 mM or less, 41 mM or less, 40 mM or less, 38 mM or less, 36 mM or less, 34 mM or less, 32 mM or less, 30 mM or less, 28 mM or less, or 26 mM or less, but is not particularly limited. ..
  • the alkali concentration is, for example, 1-50 mM, 2-50 mM, 3-50 mM, 4-50 mM, 5-45 mM, 8-45 mM, 8-50 mM, 10-45 mM, 10-50 mM, 15-45 mM, It may be 15 to 50 mM, 20 to 45 mM, 20 to 50 mM and the like.
  • the alkalinity is high, although not particularly limited. It is usually preferable to use an alkaline solution having an alkaline concentration, and for example, an alkaline solution having an alkaline concentration of about 1 to 50 mM, more preferably about 5 to 25 mM may be used.
  • the alkaline solution may or may not be used in combination with one or more surfactants, reducing agents, chelating agents, and metal salts.
  • a nucleic acid amplification reaction may be carried out after destroying the virus envelope with a pretreatment liquid containing a surfactant in advance.
  • the enveloped RNA virus in a sample can be detected with sufficient sensitivity without pretreatment with a reagent containing such a surfactant.
  • RNA virus to be tested in the present invention is not particularly limited as long as it is an RNA virus having an envelope derived from a lipid bilayer membrane.
  • enveloped RNA viruses include coronavirus family viruses (eg, SARS coronavirus, MERS coronavirus, SARS-nCOV-2 coronavirus); flaviviridae virus (eg, hepatitis C virus, Japanese encephalitis virus, deer).
  • Virus eg., ruin virus, Chikungunia virus
  • Orthomixovirus family virus eg, influenza virus
  • Rabdovir family virus eg, mad dog disease virus
  • Bunya virus family virus eg, Crimea Congo fever
  • Paramyxoviridae virus eg, measles virus, human RS virus
  • Phylloviridae virus eg, Ebola virus
  • SARS severe Acute Respiratory Syndrome
  • MERS Middle East Respiratory Syndrome
  • SARS-nCOV-2 coronavirus SARS-nCOV-2 coronavirus
  • Examples of the sample used in the present invention include pharyngeal swab, nasal swab, sputum, feces (excreted stool, rectal stool), 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 pharyngeal swabs, nasal swabs, sputum, saliva, lung aspirates, and feces (excreted stools, rectal stools).
  • samples contain proteases, nucleic acid-degrading enzymes, and the like as impurities, and feces are characterized by containing a large amount of proteins and nucleic acids derived from Escherichia coli. It is known that reaction solution components such as enzymes, primers and nucleic acid probes used in the RT-PCR reaction are digested or inactivated due to the influence of impurities contained in the sample, and the detection sensitivity is lowered. ..
  • One of the features of the present invention is that it is not necessary to perform pretreatment such as isolating / extracting RNA from these samples with a commercially available RNA purification kit or the like.
  • a nucleic acid amplification reaction may be carried out after exposing the nucleic acid of the virus by performing heat treatment or the like in advance. From the viewpoint of testing the enveloped RNA virus more easily and in a short time, in a preferred embodiment of the present invention, it is not necessary to perform prior heat treatment on such a sample.
  • These samples may be subjected to direct detection, or samples in which the sample is suspended in water, saline or buffer to reduce the effect of contaminants on the reaction and to obtain more stable test results. It may be. Further, for a sample having a particularly large amount of contaminants such as feces, the sample may be centrifuged and the supernatant may be used. 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.
  • 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 culture supernatant after isolation culture and the cultured cells contain a virus, it can be used as a sample in the present invention.
  • Examples of the types of cells used for isolation culture include MDCK cells, hCK cells, VeroE6 / TMPRSS2 cells, CHO cells, HEK-293 cells, BHK-21 cells, Sf9 cells, Sf21 cells and the like, but are particularly limited. It is not a thing, and a method similar to this is widely included.
  • 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 it in water or a buffer solution, and concentrating it with polyethylene glycol (PEG) precipitate or the like. ..
  • Specific examples of the wiping test procedure include "improvement of the norovirus test method for wiped samples" (http://idsc.nih.go.jp/iasr/32/382/dj3824.html). There is no particular limitation, and methods similar to this are widely included.
  • wipes examples 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 alkaline solution can inhibit the PCR reaction. Therefore, among the alkaline solutions, by selecting a solution having a small difference between the concentration required for denaturation of the envelope protein derived from the virus and the concentration allowed to be brought into PCR, the alkaline solution, the sample, and the 1-step RT-PCR reaction solution can be used. By adding them sequentially, the detection operation can be easily carried out in the same container from the denaturation of the envelope protein to the 1-step RT-PCR reaction without opening and closing the container in the middle.
  • the reaction vessel is opened and closed.
  • the work of opening and closing the reaction vessel becomes complicated and causes an increase in working time.
  • opening and closing the reaction vessel containing the virus-containing sample poses a 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.
  • 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, which does not have a lid opening / closing step, has more significance than simplification of the work.
  • the one-step RT-PCR solution added to the mixture comprises (i) a reverse transcriptase and a DNA polymerase, or (ii) a DNA polymerase having both reverse transcriptase activity.
  • a DNA polymerase having reverse transcription activity it is preferable to use Tth DNA polymerase, Taq DNA polymerase, or the like.
  • two enzymes for example, at least two enzymes, reverse transcriptase and DNA polymerase.
  • the origin of the reverse transcriptase (RT) 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 (Moloney Murine Leukemia Virus) -RT, AMV-RT (Avian Myeloblastosis Virus). ), HIV-RT, RAV2-RT, EIAV-RT, Carboxydothermus hydrogenoformam DNA polymerase) and variants thereof.
  • Particularly preferred examples include reverse transcriptase derived from Moloney murine leukemia virus (MMRV) (MMLV-RT), reverse transcriptase derived from avian myeloblastosis virus (AMV) (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 mutants 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 DNA polymerase during the reverse transcription reaction can be achieved by introducing it into a DNA polymerase of a thermally 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 reverse transcriptase or DNA polymerase refers to, for example, 85% or more, preferably 90% or more, based on the amino acid sequence of wild-type reverse transcriptase or wild-type DNA polymerase from which it is derived. It has a sequence identity of preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more, and has an activity of amplifying DNA in the same manner as wild reverse transcriptase or wild DNA polymerase and / /. Alternatively, it refers to an enzyme having an 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.
  • BLAST Basic local alignment search tool
  • 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 reverse transcriptase or wild-type DNA polymerase from which it is derived (hereinafter, , Collectively referred to as "mutation"), and amplifies the activity of converting RNA into DNA and / or DNA in the same manner as wild reverse transcriptase or wild DNA polymerase. It may have activity.
  • 1 or several means 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 a buffer, a magnesium salt or a manganese salt as an appropriate salt, a deoxynucleotide triphosphate, and a 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 1-step RT-PCR reaction solution 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, exhibits properties like a surfactant, and is thought 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 amount of bovine serum albumin added to the one-step RT-PCR reaction solution is not limited as long as the effects of the present invention are exhibited, but is preferably at least 0.5 mg / ml or more, and 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 1-step RT-PCR reaction solution is derived from the skin, bones, 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 that derived from fish is preferable to that derived from cattle or pig because the jelly strength is low and the reaction solution is easy to handle.
  • 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, glycerol, ethylene glycol, propylene glycol, tri.
  • EGTA Ethylene glycol-bis (2-aminoethyl ether) -N, N, N', N'-tetraacetic acid
  • BAPTA a chelating agent
  • the primer pair used in the present invention is a pair of primers corresponding to the detection 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 are used. 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, degenerate primers may be included if the target nucleic acid consists of subtypes.
  • coronavirus SARS-nCOV-2
  • a primer pair "Pathogen Detection Manual 2019-nCoV" published by the National Institute of Infectious Diseases.
  • nucleocapsid protein (N) region of SARS-nCOV-2 is detected by SEQ ID NOs: 1 and 2 and SEQ ID NOs: 4 and 5.
  • nucleocapsid (N) region, envelope protein (E) region, spike protein (S) region, RNA-dependentRNA polymerase (RdRp) region, open reading frame (ORF) Genes such as regions can be detected, but the detection is not limited to this.
  • 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 that further comprises at least one labeled hybridization probe or double-stranded DNA-bound fluorescent compound.
  • the analysis of the amplified product can be monitored by monitoring the fluorescent signal instead of the usual electrophoresis, and the analysis effort 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.
  • 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).
  • Examples of the base sequence of the probe for coronavirus detection include the sequences (SEQ ID NOs: 3 and 6) described in the "Pathogen Detection Manual 2019-nCoV” published by the National Institute of Infectious Diseases, which is also suitable for the present invention. It can be used for, but it 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.
  • genes such as N region, E region, S region, RdRp region, and ORF region can be detected, but the detection is not limited to this. No.
  • 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 further preferably 0.02 ⁇ M or more and 0.5 ⁇ M or less.
  • kits for testing viral RNA in a sample which is a pretreatment solution containing an alkaline solution, and reverse transcriptase and DNA polymerase (or DNA polymerase having reverse transcription activity), one step.
  • a kit for testing enveloped RNA virus which comprises an RT-PCR reaction solution.
  • the virus testing kit of the present invention contains at least a reagent containing an alkaline solution, reverse transcriptase, DNA polymerase, and a one-step RT-PCR reaction solution. Further, the reagent containing an alkaline solution may be a reagent containing no surfactant.
  • 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 virus test kit of the present invention can be used to test for the presence or absence of enveloped virus in a sample that has not been subjected to the separation and purification treatment of nucleic acid. Since the virus kit of the present invention can be suitably used in the virus test method of the present invention as described above, for example, the virus test kit of the present invention is a sample that has not been subjected to nucleic acid separation and purification treatment. After 1 minute or more has passed from the preparation of the mixed solution with the alkaline solution having a pH of 8 or more, the 1-step RT-PCR reaction solution is added, the reaction vessel is sealed, and then the 1-step RT-PCR reaction is carried out. obtain.
  • Such a virus test kit of the present invention can be used for testing a virus having an arbitrary envelope, but is preferably used as a coronavirus test kit, and more preferably SARS (Severe Acute Respiratory Syndrome) corona. It can be used as a test kit for the virus, MERS (Middle East Respiratory Syndrome) coronavirus, SARS-nCOV-2.
  • SARS severe Acute Respiratory Syndrome
  • MERS Middle East Respiratory Syndrome coronavirus
  • SARS-nCOV-2 Middle East Respiratory Syndrome
  • Test example 1 Examination of pretreatment of negative fecal suspension under the condition of changing pH (1) Preparation of reaction solution Using the reaction solution having the composition shown below as the basic composition, coronavirus RNA in the reaction solution was detected by 1-step RT-PCR. Other than the primers and probes, (Norovirus detection kit G1 / G2-fast probe detection Quick Step- (Toyobo) attachment) was used. In addition, the primers and probes used were the sequences described in the "Pathogen Detection Manual 2019-nCoV", and the probes were modified with FAM as the fluorescent label and BHQ1 (Black hole quencher) as the quenching group.
  • Condition 1 10 mM acetate-sodium acetate buffer (pH 5.0)
  • Condition 2 10 mM Tris-hydrochloric acid buffer (pH 7.0)
  • Condition 3 10 mM Tris-hydrochloric acid buffer (pH 8.0)
  • Condition 4 10 mM Tris-hydrochloric acid buffer (pH 9.0)
  • Condition 5 10 mM glycine-sodium hydroxide buffer (pH 9.5)
  • Sample pretreatment 1 ⁇ L of each pretreatment solution prepared in (2) is mixed with 1 ⁇ L of negative fecal suspension and allowed to stand at room temperature (25 ° C.) for 3 minutes, or immediately moved to the next step. Study was carried out.
  • RT-PCR reaction conditions This was subjected to a real-time PCR reaction using Roche's Light Cycler 96 in the following temperature cycle. 42 ° C for 10 minutes (reverse transfer conditions) 95 ° C for 1 minute (heat denaturation) 98 ° C 15 seconds-55 ° C 15 seconds-60 ° C 45 seconds 50 cycles (PCR-fluorescence reading) (6) Result
  • the Ct value was calculated by the Light Cycler 96 analysis software manufactured by Roche, and the condition calculated when the Ct value was less than 50 was regarded as positive.
  • the pH condition of the pretreatment liquid was pH 8.0 or higher, and the detection became possible by leaving the standing time for 3 minutes.
  • SARS-nCOV-2 can be detected with sufficient sensitivity by a one-step RT-PCR reaction using a sample containing a large amount of impurities that inhibit the RT-PCR reaction when mixed with an alkaline solution. It has been shown.
  • Test example 2 Examination of elapsed time after mixing with alkaline solution (1) Preparation of reaction solution Using the reaction solution having the composition shown below as the basic composition, coronavirus RNA in the reaction solution was detected by 1-step RT-PCR. Other than the primers and probes, (Norovirus detection kit G1 / G2-fast probe detection Quick Step- (Toyobo) attachment) was used. In addition, the primers and probes used were the sequences described in the "Pathogen Detection Manual 2019-nCoV", and the probes were modified with FAM as the fluorescent label and BHQ1 (Black hole quencher) as the quenching group.
  • RNAse freewater 4 ⁇ L
  • Specimen pretreatment and addition of template RNA 1 ⁇ L of the negative fecal suspension was mixed with 1 ⁇ L of the pretreatment solution (10 mM Tris-hydrochloric acid buffer (pH 8.0)), and the mixture was allowed to stand at room temperature (25 ° C.) for 0 to 3 minutes.
  • RNA coronavirus N1 RNA (Japan Genetic Research Institute: SARS-nCOV-2 RNA) was mixed with each pretreated mixture so as to have a final concentration of 50 copies / reaction, and RT- prepared in (1). 47 ⁇ L of PCR reaction solution was added, and RT-PCR was performed as a 50 ⁇ L reaction system. All the operations up to this point were carried out in the same container, and after adding the RT-PCR reaction solution, the container was sealed and then the one-step RT-PCR reaction was carried out without opening or closing the lid. (3) RT-PCR reaction conditions This was subjected to a real-time PCR reaction using Roche's Light Cycler 96 in the following temperature cycle.
  • the Ct value was calculated by the Light Cycler 96 analysis software manufactured by Roche, and the condition calculated when the Ct value was less than 50 was regarded as positive. As a result, it was confirmed that SARS-nCOV-2RNA became detectable after at least 1 minute had passed in the state of a mixed solution with the pretreatment solution.
  • Test example 3 Examination of detection in the presence of other biological specimens (1) Preparation of reaction solution Using the reaction solution having the composition shown below as the basic composition, coronavirus RNA in the reaction solution was detected by 1-step RT-PCR. Other than the primers and probes, (Norovirus detection kit G1 / G2-fast probe detection Quick Step- (Toyobo) attachment) was used. In addition, the primers and probes used were the sequences described in the "Pathogen Detection Manual 2019-nCoV", and the probes were modified with FAM as the fluorescent label and BHQ1 (Black hole quencher) as the quenching group. RT-PCR reaction solution (47 ⁇ L) Condition 1.
  • N area 1 Reaction solution 30 ⁇ L
  • Enzyme solution 5 ⁇ L 10 ⁇ M N_Sarbeco_F1 (primer, SEQ ID NO: 1): 3 ⁇ L 10 ⁇ M N_Sarbeco_R1 (primer, SEQ ID NO: 2): 4 ⁇ L 10 ⁇ M N_Sarbeco_P1 (probe, SEQ ID NO: 3): 1 ⁇ L RNAse freewater: 4 ⁇ L
  • Pretreatment of each sample To 1 ⁇ L of the pretreatment solution (10 mM Tris-hydrochloric acid buffer (pH 8.0)), 1 ⁇ L of each of the pharyngeal swab and the nasal swab was added and mixed.
  • Test example 4 Examination of pretreatment effect on inactivated virus under the condition of changing pH (1) Preparation of reaction solution Using the reaction solution having the composition shown below as the basic composition, the inactivated SARS-CoV-2 virus in the reaction solution was detected by 1-step RT-PCR. Other than the primers and probes, (Norovirus detection kit G1 / G2-fast probe detection Quick Step- (Toyobo) attachment) was used. In addition, the primers and probes used were the sequences described in the "Pathogen Detection Manual 2019-nCoV", and the probes were modified with FAM as the fluorescent label and BHQ1 (Black hole quencher) as the quenching group.
  • Condition 1 10 mM Tris-hydrochloric acid buffer (pH 8.0)
  • Condition 2 10 mM Tris-hydrochloric acid buffer (pH 8.5)
  • Condition 3 10 mM Tris-hydrochloric acid buffer (pH 9.0)
  • Condition 4 10 mM glycine-sodium hydroxide buffer (pH 9.5)
  • Pretreatment of inactivated virus 1 ⁇ L of each pretreatment solution is mixed with 1 ⁇ L of inactivated SARS-nCOV-2 virus (Inactivated SARS-CoV-2 (2019-nCoV / USA-WA1 / 2020) (ATCC)) prepared to be 10 copies / ⁇ L. Then, the mixture was allowed to stand at room temperature (25 ° C.) for 1 minute.
  • the negative control was 1 ⁇ L of RNAse free water. 48 ⁇ L of the RT-PCR reaction solution prepared in (1) was added, and RT-PCR was carried out as a 50 ⁇ L reaction system. All the operations up to this point were carried out in the same container, and after adding the RT-PCR reaction solution, the container was sealed and then the one-step RT-PCR reaction was carried out without opening or closing the lid. (4) RT-PCR reaction conditions This was subjected to a real-time PCR reaction using CFX96 DEEP WELL manufactured by Bio-Rad in the following temperature cycle.
  • Test example 5 Examination of detection in the presence of other biological specimens (1) Preparation of reaction solution Using the reaction solution having the composition shown below as the basic composition, the inactivated SARS-CoV-2 virus in the reaction solution was detected by 1-step RT-PCR. Other than the primers and probes, (Norovirus detection kit G1 / G2-fast probe detection Quick Step- (Toyobo) attachment) was used. In addition, the primers and probes used were the sequences described in the "Pathogen Detection Manual 2019-nCoV", and the probes were modified with FAM as the fluorescent label and BHQ1 (Black hole quencher) as the quenching group.
  • Saliva was collected directly in a plastic container, 100 ⁇ L of saliva and 100 ⁇ L of PBS were mixed in equal amounts, and then centrifuged (15000 rpm, 5 minutes) to collect the supernatant, which was used as a sample.
  • the pharyngeal and nasal swabs were collected from the relevant sites using a swab and suspended in a UTM virus transport medium (Copan).
  • the negative control of the pretreatment solution was 2 ⁇ L of RNAse free water.
  • 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 purpose of the present invention is to provide a method and a kit whereby the presence of an enveloped RNA virus (for example, a coronavirus) in a sample can be detected easily in a short amount of time. Provided is a method for testing an enveloped RNA virus in a sample, said method being characterized by including the following steps (1) to (3): (1) a step in which a sample that has not been subjected to nucleic acid separation/purification treatment is mixed with a reagent containing an alkaline solution; (2) a step in which a one-step RT-PCR reaction solution containing (i) a reverse transcriptase and a DNA polymerase or (ii) a DNA polymerase having reverse transcription activity is added to the liquid mixture described above at least one minute after preparing the liquid mixture; and (3) a step in which the reaction container is sealed and a one-step RT-PCR reaction is then performed.

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処理をすることなく、試料をアルカリ性溶液と混合後、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、エンベロープを持つＲＮＡウイルスの検出に関する。更に具体的には、コロナウイルスの検出法に関する。本発明により、例えば、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コロナ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 a virus by nucleic acid amplification. More specifically, RNA having an envelope is obtained by mixing the sample with an alkaline solution and then adding a reaction solution for real-time reverse transcription-polymerase chain reaction (qRT-PCR) without isolating and purifying the nucleic acid from the sample. Regarding virus detection. More specifically, it relates to a method for detecting a coronavirus. According to the present invention, it is possible to detect coronavirus contained in, for example, pharyngeal swab, nasal swab, biological sample such as sputum, fecal sample, blood sample, environmental wiping sample, etc. with high sensitivity. .. The present invention can be used for life science research, clinical diagnosis, food hygiene inspection, environmental inspection, and the like.


　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 Severe Acute Respiratory Syndrome (COVID-19) coronavirus (SARS). It is known that those causing serious fatal 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）。
For pathogen testing of coronavirus, methods for detecting viral genes using electron microscopy, ELISA-based immunological antigen detection, or nucleic acid amplification techniques have 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は煩雑であり、多くの作業時間を要していた。
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). In Japan as well, 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 process of extracting and purifying viral RNA was complicated and required a lot of working time.


　これまでに、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、糞便試料中では多糖類などのＰＣＲ反応阻害物質が持ち込まれる。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。そして未だ、コロナウイルス、特にＳＡＲＳ－ｎＣＯＶ－２を含む咽頭・鼻腔ぬぐい液や糞便試料などの生体試料やふき取り環境試料からＲＴ－ＰＣＲ反応により検査を行う方法において、ＲＮＡの分離精製工程を必要とせず、簡便に、短時間でこれらのウイルスを検出できる手法は知られていない。更に可能な限り作業工程を減らして簡略化することで、検査を行う医療従事者への感染リスクを低減させ、コロナウイルス、特に、ＳＡＲＳ－ｎＣＯＶ－２を検査する手法の開発が強く求められている。
So far, K.K. Kang et al. Report that highly pathogenic North American pig genital respiratory syndrome viral RNA can be detected directly from porcine 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 widely depending on the type of sample. For example, PCR reaction inhibitors such as polysaccharides are introduced into stool samples. In addition, it is known that virus inactivation and RNA extraction conditions differ greatly depending on the virus species. And still, RNA separation and purification steps are required in the method of testing by RT-PCR reaction from biological samples such as pharyngeal / nasal swabs and fecal samples containing coronavirus, especially SARS-nCOV-2, and wiping environment samples. However, there is no known method that can detect these viruses easily and in a short time. Furthermore, by reducing and simplifying the work process as much as possible, the risk of infection to medical staff performing the test is reduced, and there is a strong demand for the development of a method for testing coronavirus, especially SARS-nCOV-2. There is.


特開２０１２－２４０３９号公報Japanese Unexamined Patent Publication No. 2012-24039


ＪＯＵＲＮＡＬ　ＯＦ　ＣＬＩＮＩＣＡＬ　ＭＩＣＲＯＢＩＯＬＯＧＹ，Ｎｏｖ．２００５，ｐ．５４５２-５４５６JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2005, p. 5452-5456 Ｊ Ｖｉｒｏｌ　Ｍｅｔｈｏｄｓ．　２００４　Ｓｅｐ　１；１２０（１）：３３－４０．J Virol Methods. 2004 Sep 1; 120 (1): 33-40. Ｐｕｂｌｉｓｈｅｄ　Ｏｎｌｉｎｅ　Ｊａｎｕａｒｙ ２９，２０２０，ｈｔｔｐｓ：／／ｄｏｉ．ｏｒｇ／１０．１０１６／Ｓ０１４０－６７３６（２０）３０２５１－８Published Online January 29, 2020, https: // doi. org / 10.1016 / S0140-6736 (20) 30251-8 世界保健機関（ＷＨＯ）ホームページ（Ｄｉａｇｎｏｓｔｉｃ　ｄｅｔｅｃｔｉｏｎ　ｏｆ　Ｗｕｈａｎ　ｃｏｒｏｎａｖｉｒｕｓ　２０１９　ｂｙ　ｒｅａｌ－ｔｉｍｅＲＴ－ＰＣＲ）World Health Organization (WHO) homepage (Diagnostic detection of Wuhan coronavirus 2019 by real-timeRT-PCR) 国立感染症研究所ホームページ「病原体検出マニュアル２０１９－ｎＣｏＶ 」（ｈｔｔｐｓ：／／ｗｗｗ．ｎｉｉｄ．ｇｏ．ｊｐ／ｎｉｉｄ／ｉｍａｇｅｓ／ｌａｂ－ｍａｎｕａｌ／２０１９－ｎＣｏＶ２０２００２１７．ｐｄｆ）National Institute of Infectious Diseases Homepage "Pathogen Detection Manual 2019-nCoV" (https://www.niid.go.jp/niid/images/lab-manual/2019-nCoV20200217.pdf) Ｊ．Ａｎｉｍａｌ　Ｓｃｉｅｎｃｅ　Ａｎｄ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，第５巻、２０１４年、第４５頁J. Animal Science And Biotechnology, Vol. 5, 2014, p. 45


　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、試料から事前に核酸の分離や精製を行うことなく、簡便に、短時間で、１ステップＲＴ－ＰＣＲにより、エンベロープを持つウイルス、特にコロナウイルス（なかでも、ＳＡＲＳ－ｎＣＯＶ－２）のＲＮＡの有無の検出を可能とすることである。
The present invention has been made against the background of the problems of the prior art. That is, RNA of enveloped viruses, especially coronavirus (especially SARS-nCOV-2), can be easily and quickly performed by 1-step RT-PCR without separating or purifying nucleic acids from the sample in advance. It is possible to detect the presence or absence of virus.


　本発明者らは、上記事情に鑑み、鋭意研究を行った結果、事前に核酸の分離精製を行っていない試料をアルカリ性溶液と混合後、１ステップＲＴ－ＰＣＲに供することで、試料中に含まれるエンベロープを持つウイルス、特にコロナウイルス（なかでも、ＳＡＲＳ－ｎＣＯＶ－２）を十分な感度で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 found that a sample in which nucleic acid has not been separated and purified in advance is mixed with an alkaline solution and then subjected to 1-step RT-PCR to be contained in the sample. We have found that a virus having an envelope, particularly a coronavirus (among others, SARS-nCOV-2), can be detected with sufficient sensitivity, and arrived at the present invention.


代表的な本願発明は、以下の通りである。

項１.　試料中のエンベロープを持つ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）核酸の分離精製処理を行っていない試料とｐＨ８以上のアルカリ性溶液とを混合する工程、

（２）前記混合液の調製から１分以上経過した後に、（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、

（３）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.　前記工程（１）～（３）が同一容器で行われることを特徴とする項１に記載の検査方法。

項３.　工程（３）において反応容器を密閉後、一度もフタを開閉することなく１ステップＲＴ－ＰＣＲ反応を実施することを特徴とする項１又は２に記載の検査方法。

項４.アルカリ性溶液が、水酸化カリウム水溶液、水酸化ナトリウム水溶液、水酸化リチウム水溶液、水酸化マグネシウム水溶液、水酸化カルシウム水溶液、水酸化バリウム水溶液、炭酸カリウム水溶液、炭酸ナトリウム水溶液、炭酸マグネシウム水溶液、炭酸カルシウム水溶液、トリス緩衝液、グリシン緩衝液、リン酸緩衝液、ホウ酸緩衝液、又はグッド緩衝液である項１から３のいずれかに記載の検査方法。

項５.　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、及び培養上清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４のいずれかに記載の検査方法。

項６.　試料が、水、生理食塩水または緩衝液に予め懸濁された懸濁液である項１から５のいずれかに記載の検査方法。

項７.　試料が、懸濁液の遠心上清である項６に記載の検査方法。

項８.  試料が、環境中の拭き取り検査試料を水、生理食塩水または緩衝液に予め懸濁し、かつ、該懸濁液を濃縮した試料である項１から７のいずれかに記載の検査方法。

項９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１から８のいずれかに記載の検査方法。

項１０.　コロナウイルスが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、又はＳＡＲＳ－ｎＣＯＶ－２コロナウイルスである項９に記載の検査方法。

項１１.　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少なくとも1種である項１から１０のいずれかに記載の検査方法。

項１２.　逆転写酵素が、モロニーマウス白血病ウイルス（ＭＭＲＶ）由来逆転写酵素、トリ骨髄芽球症ウイルス（ＡＭＶ）由来逆転写酵素、およびこれらの変異体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１１のいずれかに記載の検査方法。

項１３.　工程（３）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つを含む項１から１２のいずれかに記載の検査方法。

項１４.　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１３に記載の検査方法。

項１５.　ｐＨ８以上のアルカリ性溶液、逆転写酵素、ＤＮＡポリメラーゼ、および１ステップＲＴ－ＰＣＲ反応液を含むことを特徴とするエンベロープを持つウイルスの検査用キット。

項１６.  核酸の分離精製処理を行っていない試料から、エンベロープを持つウイルスの有無を検査するために用いられる、項１５に記載のウイルスの検査用キット。

項１７.　核酸の分離精製処理を行っていない試料とｐＨ８以上のアルカリ性溶液との混合液の調製から１分以上経過した後に、１ステップＲＴ－ＰＣＲ反応液を添加し、反応容器を密閉後、１ステップＲＴ－ＰＣＲ反応を実施するようにして用いられる、項１６に記載のウイルスの検査用キット。

項１８　アルカリ性溶液が、水酸化カリウム水溶液、水酸化ナトリウム水溶液、水酸化リチウム水溶液、水酸化マグネシウム水溶液、水酸化カルシウム水溶液、水酸化バリウム水溶液、炭酸カリウム水溶液、炭酸ナトリウム水溶液、炭酸マグネシウム水溶液、炭酸カルシウム水溶液、トリス緩衝液、グリシン緩衝液、リン酸緩衝液、ホウ酸緩衝液、又はグッド緩衝液である、項１５から１７のいずれかに記載のウイルスの検査用キット。

項１９.　１ステップＲＴ－ＰＣＲ反応液が、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つを含む項１５から１８のいずれかに記載のウイルスの検査用キット。

項２０.　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１５から１９のいずれかに記載のウイルスの検査用キット。

項２１.　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１５から２０のいずれかに記載のウイルスの検査用キット。

項２２.　エンべロープを持つＲＮＡウイルスがコロナウイルスである項１５から２１のいずれかに記載のウイルスの検査用キット。

項２３.　コロナウイルスが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、又はＳＡＲＳ－ｎＣＯＶ－２であることを特徴とする項２２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 testing an RNA virus having an envelope in a sample, which comprises the following steps:

(1) A step of mixing a sample that has not been separated and purified from nucleic acid with an alkaline solution having a pH of 8 or higher.

(2) 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 after 1 minute or more has passed from the preparation of the mixed solution.

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

Item 2. The inspection method according to Item 1, wherein the steps (1) to (3) are performed in the same container.

Item 3. The inspection method according to Item 1 or 2, wherein the reaction vessel is sealed in the step (3), and then the one-step RT-PCR reaction is carried out without opening or closing the lid.

Item 4. The alkaline solution is potassium hydroxide aqueous solution, sodium hydroxide aqueous solution, lithium hydroxide aqueous solution, magnesium hydroxide aqueous solution, calcium hydroxide aqueous solution, barium hydroxide aqueous solution, potassium carbonate aqueous solution, sodium carbonate aqueous solution, magnesium carbonate aqueous solution, carbon dioxide. Item 4. The test method according to any one of Items 1 to 3, which is an aqueous calcium solution, a Tris buffer solution, a glycine buffer solution, a phosphate buffer solution, a borate buffer solution, or a good buffer solution.

Item 5. At least one sample selected from the group consisting of feces, pharyngeal swab, nasal swab, sputum, pulmonary aspirate, cerebrospinal fluid, mouthwash, saliva, tears, cultured cells, and culture supernatant. The inspection method according to any one of Items 1 to 4, which is a species.

Item 6. The test method according to any one of Items 1 to 5, wherein the sample is a suspension previously suspended in water, physiological saline or a buffer solution.

Item 7. The inspection method according to Item 6, wherein the sample is a centrifugal supernatant of a suspension.

Item 8. The inspection method according to any one of Items 1 to 7, wherein the sample is a sample in which an environmental wiping test sample is previously suspended in water, physiological saline or a buffer solution, and the suspension is concentrated. ..

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

Item 10. The test method according to Item 9, wherein the coronavirus is SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, or SARS-nCOV-2 coronavirus.

Item 11. The test method according to any one of Items 1 to 10, wherein the DNA polymerase is at least one selected from the group consisting of Taq, Tth and variants thereof.

Item 12. The reverse transcriptase is at least one selected from the group consisting of Moloney murine leukemia virus (MMRV) -derived reverse transcriptase, avian myeloblastosis virus (AMV) -derived reverse transcriptase, and variants thereof. The inspection method according to any one of Items 1 to 11.

Item 13. The 1-step RT-PCR reaction solution in step (3) has a structure in which three methyl groups are added to an amino group in an amino acid, and is a quaternary ammonium salt (hereinafter referred to as “betaine-like quaternary ammonium”). The test method according to any one of Items 1 to 12, which comprises at least one selected from the group consisting of bovine serum albumin, glycerol, glycol and gelatin.

Item 14. The inspection method according to Item 13, wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine.

Item 15. A kit for testing a virus having an envelope, which comprises an alkaline solution having a pH of 8 or higher, a reverse transcriptase, a DNA polymerase, and a 1-step RT-PCR reaction solution.

Item 16. The virus inspection kit according to Item 15, which is used for inspecting the presence or absence of an enveloped virus from a sample that has not been subjected to the separation and purification treatment of nucleic acid.

Item 17. After 1 minute or more has passed from the preparation of the mixed solution of the sample not subjected to the separation and purification treatment of the nucleic acid and the alkaline solution having a pH of 8 or more, the 1-step RT-PCR reaction solution is added, the reaction vessel is sealed, and then the reaction vessel is sealed. Item 6. The virus testing kit according to Item 16, which is used to carry out a one-step RT-PCR reaction.

Item 18 The alkaline solution is potassium hydroxide aqueous solution, sodium hydroxide aqueous solution, lithium hydroxide aqueous solution, magnesium hydroxide aqueous solution, calcium hydroxide aqueous solution, barium hydroxide aqueous solution, potassium carbonate aqueous solution, sodium carbonate aqueous solution, magnesium carbonate aqueous solution, calcium carbonate. Item 6. The virus test kit according to any one of Items 15 to 17, which is an aqueous solution, a Tris buffer solution, a glycine buffer solution, a phosphate buffer solution, a borate buffer solution, or a Good buffer solution.

Item 19. The one-step RT-PCR reaction solution according to any one of Items 15 to 18, which comprises at least one selected from the group consisting of betaine-like quaternary ammonium salt, bovine serum albumin, glycerol, glycol and gelatin. Virus test kit.

Item 20. The virus testing kit according to any one of Items 15 to 19, further comprising a primer pair corresponding to the detection region of the RNA virus to be detected.

Item 21. The virus testing kit according to any one of Items 15 to 20, further comprising a hybridization probe corresponding to the detection region of the RNA virus to be detected.

Item 22. The virus test kit according to any one of Items 15 to 21, wherein the RNA virus having an envelope is a coronavirus.

Item 23. The virus test kit according to Item 22, wherein the coronavirus is SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, or SARS-nCOV-2. ..


　本発明によって、試料から事前の核酸の分離精製処理を必要とせず、試料をアルカリ性溶液と混合後、１ステップＲＴ－ＰＣＲ反応液に添加するだけで、試料中のコロナウイルスのようなエンベロープウイルスの有無を簡便に且つ短時間に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、感染症の早期発見や感染拡大防止にも寄与する。また、ウイルスＲＮＡの精製工程の省略化により、反応容器の蓋の開閉作業も省略される。この結果、蓋の開閉時におけるウイルス含有サンプルの飛散リスクをなくすことができ、他のサンプルへのコンタミリスクも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、ウイルス含有サンプルの飛散リスクをなくす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, it is not necessary to separate and purify the nucleic acid from the sample in advance, and the sample is mixed with an alkaline solution and then added to the 1-step RT-PCR reaction solution to obtain an enveloped virus such as coronavirus in the sample. The presence or absence can be easily detected in a short time. As a result, the efficiency of inspection work can be further improved, so that the number of inspections can be increased, which contributes to early detection of infectious diseases and prevention of spread of infection. Further, by omitting the step of purifying the viral RNA, the work of opening and closing the lid of the reaction vessel is also omitted. As a result, the risk of scattering of the virus-containing sample when opening and closing the lid can be eliminated, and the risk of contamination to other samples can also 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 workers can be reduced by eliminating the risk of scattering the virus-containing sample.


　特に、２０１９年に発生したＳＡＲＳ－ｎＣＯＶ－２（ＳＡＲＳ－ＣｏＶ－２、２０１９－ｎＣｏＶ等とも呼称される場合がある）を含みうる試料の検査において、本発明は優れた効果を奏する。例えば、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（特に、ＳＡＲＳ－ｎＣＯＶ－２）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。
In particular, the present invention exerts an excellent effect in the inspection of a sample that may contain SARS-nCOV-2 (which may also be referred to as SARS-CoV-2, 2019-nCoV, etc.) that occurred in 2019. For example, according to the present invention, blood, feces (excretion, rectal stool), vomitus, urine, sputum, lymph, plasma, ejaculation, pulmonary aspirate, cerebrospinal fluid, pharyngeal swab, nasal swab, gargling. High sensitivity of coronavirus (particularly SARS-nCOV-2) from contaminant-rich samples such as biological samples containing fluids, saliva, tears, environmental wipes, cultured cells or samples containing culture supernatants. Can also be detected. The present invention can also be used for life science research, clinical diagnosis, food hygiene inspection, environmental inspection, and the like.


　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 specified.

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". 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 stated.


　本発明の一態様は、試料中のコロナウイルス（特に、ＳＡＲＳ－ｎＣＯＶ－２）などのエンベロープを持つＲＮＡウイルス（本明細書では、これを「エンベローブＲＮＡウイルス」等ともいう）の検査方法であって、試料から事前にウイルスＲＮＡの分離精製を実施することなく、試料とアルカリ性溶液を含む試薬とを混合し、（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試薬を添加することを包含する、エンベロープを持つＲＮＡウイルスの有無を検査するための方法である。
One aspect of the present invention is a method for testing an RNA virus having an envelope such as coronavirus (particularly SARS-nCOV-2) in a sample (hereinafter, this is also referred to as "emberobe RNA virus" or the like). Then, without performing separation and purification of viral RNA from the sample in advance, the sample and a reagent containing an alkaline solution are mixed to obtain (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcription activity. A method for examining the presence or absence of an enveloped RNA virus, comprising adding a one-step RT-PCR reagent comprising.


　本発明の試料中のエンベロープを持つＲＮＡウイルスの有無を検査するための方法は、少なくとも以下の工程が含まれることを特徴とする検査方法である。

（１）核酸の分離精製処理を行っていない試料とアルカリ性溶液を含む試薬とを混合する工程、

（２）前記混合液の調製から１分以上経過した後に、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、

（３）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）および（２）、好ましくは工程（１）～（３）の全ては、同一容器で行われることが好ましい。すなわち、工程（１）および（２）の間、好ましくは工程（１）～（３）の各工程の間においては、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）により混合液の全量を工程（２）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（２）を実施しても良い。更には、工程（３）においては、反応容器を密閉後、反応容器の蓋の開閉を行わないことが好ましい。
The method for inspecting the presence or absence of an enveloped RNA virus in a sample of the present invention is an inspection method comprising at least the following steps.

(1) A step of mixing a sample that has not been separated and purified from nucleic acid with a reagent containing an alkaline solution.

(2) After 1 minute or more has passed from the preparation of the mixed solution, a 1-step RT-PCR reaction solution containing (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcription activity is added to the mixed solution. Addition process,

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

It is preferable that all of the steps (1) and (2), preferably steps (1) to (3), 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 (2), preferably between the steps (1) to (3). The entire amount of the mixed solution may be subjected to the step (2) according to the step (1), or a part thereof may be transferred to another container to carry out the step (2). Further, in the step (3), it is preferable not to open or close the lid of the reaction vessel after sealing the reaction vessel.


　本発明は、工程（１）にて得た混合液を、１分以上混合状態のままで反応させることを一つの特徴とする。このように、核酸の分離精製処理を行っていない試料とアルカリ性溶液を含む試薬とを混合して１分以上反応させることによって、採取した生体試料中に夾雑物などのＰＣＲ反応阻害物質が含まれている場合であっても、１ステップＲＴ－ＰＣＲ反応において、その試料中に含まれ得るエンベロープを持つウイルス（例えば、コロナウイルス、なかでもＳＡＲＳ－ｎＣＯＶ－２）を検出することが可能となる。前記混合液調製後の反応時間は、１分以上であれば、本発明の効果を奏する限り特に限定されない。例えば、工程（２）にて１分間～２０分間程度経過後に１ステップＲＴ－ＰＣＲ反応液の添加を実施してもよいし、２０分間以上経過後に１ステップＲＴ－ＰＣＲ反応を実施してもよい。より短時間でエンベロープＲＮＡウイルスの検査を行うという観点から、好ましくは、工程（１）で混合液を得た後、工程（２）にてＲＴ－ＰＣＲ反応液の添加までの時間が１分以上１０分以下であることが好ましく、１分以上５分以下であることがより好ましく、１分以上４分以下であることが更に好ましく、１分以上３分以下であることが特に好ましい。また、工程（２）のＲＴ－ＰＣＲ反応液を添加するまでの混合液での状態は、静置状態でもよいし、ボルテックスミキサーや分注機等を利用した攪拌状態においてもよい。
One of the features of the present invention is that the mixed solution obtained in the step (1) is reacted in a mixed state for 1 minute or longer. In this way, by mixing a sample that has not been separated and purified of nucleic acid and a reagent containing an alkaline solution and reacting for 1 minute or more, a PCR reaction inhibitor such as a contaminant is contained in the collected biological sample. Even if this is the case, it is possible to detect a virus having an envelope (for example, coronavirus, especially SARS-nCOV-2) that can be contained in the sample in the one-step RT-PCR reaction. The reaction time after preparing the mixed solution is not particularly limited as long as it is 1 minute or more as long as the effects of the present invention are exhibited. For example, the addition of the 1-step RT-PCR reaction solution may be carried out after a lapse of about 1 to 20 minutes in the step (2), or the 1-step RT-PCR reaction may be carried out after a lapse of 20 minutes or more. .. From the viewpoint of testing for enveloped RNA virus in a shorter time, it is preferable that the time from obtaining the mixed solution in step (1) to adding the RT-PCR reaction solution in step (2) is 1 minute or more. It is preferably 10 minutes or less, more preferably 1 minute or more and 5 minutes or less, further preferably 1 minute or more and 4 minutes or less, and particularly preferably 1 minute or more and 3 minutes or less. Further, the state of the mixed solution until the RT-PCR reaction solution of the step (2) is added may be a stationary state or a stirring state using a vortex mixer, a dispenser or the like.


　本発明において、工程（２）にてＲＴ－ＰＣＲ反応液を添加するまでの、工程（１）にて得た混合液の温度条件は特に制限されない。混合液が凍結しない程度の低温（例えば、－１０℃～１０℃程度（好ましい例として、氷上など））、室温（例えば、１０℃～４０℃程度）、高温（４０℃以上）などの温度域が挙げられる。好ましくは低温又は室温において反応させるのがよく、より好ましくは氷上～室温の温度条件下で反応させるのがよく、作業性も考慮すると、好ましくは室温において反応させるのがよい。
In the present invention, the temperature conditions of the mixed solution obtained in the step (1) until the RT-PCR reaction solution is added in the step (2) are not particularly limited. Temperature range such as low temperature (for example, about -10 ° C to 10 ° C (preferably on ice)), room temperature (for example, about 10 ° C to 40 ° C), high temperature (40 ° C or higher) so that the mixed solution does not freeze. Can be mentioned. The reaction is preferably carried out at a low temperature or room temperature, more preferably under temperature conditions of ice to room temperature, and in consideration of workability, the reaction is preferably carried out at room temperature.


　本発明において、試料又はその懸濁液若しくは溶解液とアルカリ性溶液とを混合する方法は特に制限されないが、例えばＲＴ－ＰＣＲ反応容器内にあらかじめ分注されたアルカリ性溶液に、分注機等を利用して試料を添加し混合する方法、試料の付着した採取具、例えばスワブや採便器具をアルカリ性溶液に直接接触させて懸濁する方法などが挙げられる。
In the present invention, the method of mixing the sample or its suspension or solution with the alkaline solution is not particularly limited, and for example, a dispenser or the like is used for the alkaline solution previously dispensed in the RT-PCR reaction vessel. Then, a method of adding and mixing the sample, a method of suspending the sampling tool to which the sample is attached, for example, a swab or a stool collecting device in direct contact with an alkaline solution, and the like can be mentioned.


　本発明において、アルカリ性溶液はアルカリ性の溶液であれば特に限定されない。アルカリ性溶液としては、例えば水酸化カリウム水溶液、水酸化ナトリウム水溶液、水酸化リチウム水溶液、水酸化マグネシウム水溶液、水酸化カルシウム水溶液、水酸化バリウム水溶液、炭酸カリウム水溶液、炭酸ナトリウム水溶液、炭酸マグネシウム水溶液、炭酸カルシウム水溶液、トリス緩衝液、グリシン緩衝液、リン酸緩衝液、ホウ酸緩衝液、グッド緩衝液（例えば、ＴＡＰＳＯ，ＰＯＰＳＯ、ＨＥＰＳＯ、ＥＰＰＳ、Ｔｒｉｃｉｎｅ、Ｂｉｃｉｎｅ、ＴＡＰＳ、ＣＨＥＳ、ＣＡＰＳ）などが挙げられ、１種単独又は２種以上組み合わせて使用できる。好ましいアルカリ性溶液は、トリス緩衝液、トリシン緩衝液、及びグリシン緩衝液からなる群より選択される少なくとも１種の液である。より好ましいアルカリ性溶液は、トリス緩衝液又はグリシン緩衝液である。
In the present invention, the alkaline solution is not particularly limited as long as it is an alkaline solution. Examples of the alkaline solution include potassium hydroxide aqueous solution, sodium hydroxide aqueous solution, lithium hydroxide aqueous solution, magnesium hydroxide aqueous solution, calcium hydroxide aqueous solution, barium hydroxide aqueous solution, potassium carbonate aqueous solution, sodium carbonate aqueous solution, magnesium carbonate aqueous solution, and calcium carbonate. Examples thereof include aqueous solution, Tris buffer solution, glycine buffer solution, phosphate buffer solution, borate buffer solution, Good buffer solution (for example, TAPSO, POPSO, HEPSO, EPPS, Tricine, Bine, TAPS, CHES, CAPS). Can be used alone or in combination of two or more. A preferred alkaline solution is at least one solution selected from the group consisting of Tris buffer, Tricine buffer, and Glycine buffer. A more preferred alkaline solution is Tris buffer or Glycine buffer.


　前記アルカリ性溶液は、アルカリ性の溶液であれば特に限定されないが、例えば、水素イオン指数（ｐＨ）（２５℃）として、ｐＨ８．０以上、ｐＨ８．５以上、ｐＨ９．０以上、ｐＨ９．５以上、ｐＨ１０．以上、ｐＨ１０．５以上、ｐＨ１１．０以上、ｐＨ１１．５以上、ｐＨ１２．０以上、ｐＨ１２．５以上、ｐＨ１３．０以上、ｐＨ１３．５以上またはｐＨ１４．０以上でありうる。より確実に高い本発明の効果が期待できるという観点から、アルカリ性溶液は、ｐＨ８～１４程度であることが好ましく、ｐＨ８～１２程度であることが更に好ましく、ｐＨ８～１０程度であることが更により好ましく、なかでもｐＨ８～９．５程度であることが好ましい。
The alkaline solution is not particularly limited as long as it is an alkaline solution, and for example, the hydrogen ion index (pH) (25 ° C.) is pH 8.0 or higher, pH 8.5 or higher, pH 9.0 or higher, pH 9.5 or higher, pH 10. As described above, the pH may be 10.5 or higher, pH 11.0 or higher, pH 11.5 or higher, pH 12.0 or higher, pH 12.5 or higher, pH 13.0 or higher, pH 13.5 or higher, or pH 14.0 or higher. From the viewpoint that a higher effect of the present invention can be expected more reliably, the alkaline solution preferably has a pH of about 8 to 14, more preferably about 8 to 12, and even more preferably about pH 8 to 10. It is preferable that the pH is about 8 to 9.5.


　本発明に用いるアルカリ性溶液は、上記ｐＨ範囲となるように調整されていれば、そのアルカリ性物質の濃度（「アルカリ濃度」ともいう）は特に限定されない。ここで、アルカリ濃度は体積モル濃度（Ｍ又はｍｏｌ／Ｌ）であり、アルカリ性溶液１Ｌ中に含まれるアルカリ性物質（例えば、上記のような水酸化カリウム及び／又は水酸化ナトリウムなどのアルカリ性物質の総量）のモル量で規定できる。
The concentration of the alkaline substance (also referred to as "alkaline concentration") of the alkaline solution used in the present invention is not particularly limited as long as it is adjusted to be within the above pH range. Here, the alkaline concentration is a volume molar concentration (M or mol / L), and the total amount of alkaline substances (for example, potassium hydroxide and / or sodium hydroxide as described above) contained in 1 L of the alkaline solution. ) Can be specified by the molar amount.


　アルカリ濃度は、例えば１ｍＭ以上、１．５ｍＭ以上、２ｍＭ以上、４ｍＭ以上、５ｍＭ以上、６ｍＭ以上、８ｍＭ以上、又は１０ｍＭ以上であり得、また、例えば５０ｍＭ以下、４９ｍＭ以下、４８ｍＭ以下、４７ｍＭ以下、４６ｍＭ以下、４５ｍＭ以下、４４ｍＭ以下、４３ｍＭ以下、４２ｍＭ以下、４１ｍＭ以下、４０ｍＭ以下、３８ｍＭ以下、３６ｍＭ以下、３４ｍＭ以下、３２ｍＭ以下、３０ｍＭ以下、２８ｍＭ以下、又は２６ｍＭ以下であり得るが、特に限定されない。
The alkali concentration can be, for example, 1 mM or more, 1.5 mM or more, 2 mM or more, 4 mM or more, 5 mM or more, 6 mM or more, 8 mM or more, or 10 mM or more, and for example, 50 mM or less, 49 mM or less, 48 mM or less, 47 mM or less, It may be 46 mM or less, 45 mM or less, 44 mM or less, 43 mM or less, 42 mM or less, 41 mM or less, 40 mM or less, 38 mM or less, 36 mM or less, 34 mM or less, 32 mM or less, 30 mM or less, 28 mM or less, or 26 mM or less, but is not particularly limited. ..


　一実施形態において、アルカリ濃度は、例えば１～５０ｍＭ、２～５０ｍＭ、３～５０ｍＭ、４～５０ｍＭ、５～４５ｍＭ、８～４５ｍＭ、８～５０ｍＭ、１０～４５ｍＭ、１０～５０ｍＭ、１５～４５ｍＭ、１５～５０ｍＭ、２０～４５ｍＭ、２０～５０ｍＭなどとしてもよい。特定の実施形態において、例えば、コロナウイルスであるＳＡＲＳ－ＣｏＶ－２等を検出するために糞便検体、咽頭ぬぐい液、鼻腔ぬぐい液を生体試料として用いる場合などには、特に限定されないが、高いアルカリ濃度のアルカリ性溶液を用いることが通常好ましく、例えば、１～５０ｍＭ程度、より好ましくは５～２５ｍＭ程度のアルカリ濃度のアルカリ性溶液を用いてもよい。
In one embodiment, the alkali concentration is, for example, 1-50 mM, 2-50 mM, 3-50 mM, 4-50 mM, 5-45 mM, 8-45 mM, 8-50 mM, 10-45 mM, 10-50 mM, 15-45 mM, It may be 15 to 50 mM, 20 to 45 mM, 20 to 50 mM and the like. In a specific embodiment, for example, when a fecal sample, a pharyngeal swab, or a nasal swab is used as a biological sample to detect SARS-CoV-2 or the like, which is a coronavirus, the alkalinity is high, although not particularly limited. It is usually preferable to use an alkaline solution having an alkaline concentration, and for example, an alkaline solution having an alkaline concentration of about 1 to 50 mM, more preferably about 5 to 25 mM may be used.


　前記アルカリ性溶液は、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、これらと組み合わせて使用しなくてもよい。ウイルスの検査方法では、事前に界面活性剤を含む前処理液でウイルスのエンベロープを破壊してから核酸増幅反応を行う場合がある。本発明では、このような界面活性剤を含む試薬を用いた前処理を行わなくても、試料中のエンベロープＲＮＡウイルスを十分な感度で検出可能である。
The alkaline solution may or may not be used in combination with one or more surfactants, reducing agents, chelating agents, and metal salts. In a virus testing method, a nucleic acid amplification reaction may be carried out after destroying the virus envelope with a pretreatment liquid containing a surfactant in advance. In the present invention, the enveloped RNA virus in a sample can be detected with sufficient sensitivity without pretreatment with a reagent containing such a surfactant.


　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ウイルスであれば、特に限定されるものではない。このようなエンベロープＲＮＡウイルスとしては、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）；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）；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）；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）；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く、特にコロナウイルス、なかでも、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コロナウイルスの検出に有用である。
The RNA virus to be tested in the present invention is not particularly limited as long as it is an RNA virus having an envelope derived from a lipid bilayer membrane. Such enveloped RNA viruses include coronavirus family viruses (eg, SARS coronavirus, MERS coronavirus, SARS-nCOV-2 coronavirus); flaviviridae virus (eg, hepatitis C virus, Japanese encephalitis virus, deer). Virus); Togavirus family virus (eg, ruin virus, Chikungunia virus); Orthomixovirus family virus (eg, influenza virus); Rabdovir family virus (eg, mad dog disease virus); Bunya virus family virus (eg, Crimea Congo fever) Virus); Paramyxoviridae virus (eg, measles virus, human RS virus); Phylloviridae virus (eg, Ebola virus), etc., but are not particularly limited, especially coronavirus, among others. It is useful for detecting SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, and SARS-nCOV-2 coronavirus.


　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、咽頭ぬぐい液、鼻腔ぬぐい液、喀痰、唾液、肺吸引物、糞便（排泄便、直腸便）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等が含まれている他、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。本発明においては、これら試料を市販のＲＮＡ精製キットなどでＲＮＡを単離・抽出する等の前処理を行う必要がないことを一つの特徴とし得る。また、一般にウイルスの検査方法では、事前に熱処理等を行ってウイルスの核酸を露出させてから核酸増幅反応を行う場合がある。より簡便に且つ短時間でエンベロープを持つＲＮＡウイルスの検査を行うという観点から、本発明の好ましい実施形態では、このような試料の事前の熱処理を行わなくてもよい。これらの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。
Examples of the sample used in the present invention include pharyngeal swab, nasal swab, sputum, feces (excreted stool, rectal stool), 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 pharyngeal swabs, nasal swabs, sputum, saliva, lung aspirates, and feces (excreted stools, rectal stools). These samples contain proteases, nucleic acid-degrading enzymes, and the like as impurities, and feces are characterized by containing a large amount of proteins and nucleic acids derived from Escherichia coli. It is known that reaction solution components such as enzymes, primers and nucleic acid probes used in the RT-PCR reaction are digested or inactivated due to the influence of impurities contained in the sample, and the detection sensitivity is lowered. .. One of the features of the present invention is that it is not necessary to perform pretreatment such as isolating / extracting RNA from these samples with a commercially available RNA purification kit or the like. In general, in a virus inspection method, a nucleic acid amplification reaction may be carried out after exposing the nucleic acid of the virus by performing heat treatment or the like in advance. From the viewpoint of testing the enveloped RNA virus more easily and in a short time, in a preferred embodiment of the present invention, it is not necessary to perform prior heat treatment on such a sample. These samples may be subjected to direct detection, or samples in which the sample is suspended in water, saline or buffer to reduce the effect of contaminants on the reaction and to obtain more stable test results. It may be. Further, for a sample having a particularly large amount of contaminants such as feces, the sample may be centrifuged and the supernatant may be used. 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.


　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 aspect 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 culture supernatant after isolation culture and the cultured cells contain a virus, it can be used as a sample in the present invention. Examples of the types of cells used for isolation culture include MDCK cells, hCK cells, VeroE6 / TMPRSS2 cells, CHO cells, HEK-293 cells, BHK-21 cells, Sf9 cells, Sf21 cells and the like, but are particularly limited. It is not a thing, and a method similar to this is widely included.


　本発明における別の態様の試料としては、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. 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 it in water or a buffer solution, and concentrating it with polyethylene glycol (PEG) precipitate or the like. .. Specific examples of the wiping test procedure include "improvement of the norovirus test method for wiped samples" (http://idsc.nih.go.jp/iasr/32/382/dj3824.html). 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.


　前記アルカリ性溶液はＰＣＲ反応を阻害しうることが知られる。そのため、前記アルカリ性溶液の中でも、ウイルス由来のエンベロープ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、アルカリ性溶液、試料、１ステップＲＴ－ＰＣＲ反応液と順次添加することで、エンベロープ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。
It is known that the alkaline solution can inhibit the PCR reaction. Therefore, among the alkaline solutions, by selecting a solution having a small difference between the concentration required for denaturation of the envelope protein derived from the virus and the concentration allowed to be brought into PCR, the alkaline solution, the sample, and the 1-step RT-PCR reaction solution can be used. By adding them sequentially, the detection operation can be easily carried out in the same container from the denaturation of the envelope protein to the 1-step RT-PCR reaction without opening and closing the container in the middle.


　試料の移し替えまたは加熱処理工程の際には、反応容器の開閉作業が生じる。反応容器の開閉作業は煩雑化かつ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開閉には、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、蓋の開閉工程のない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。
During the sample transfer or heat treatment process, the reaction vessel is opened and closed. The work of opening and closing the reaction vessel becomes complicated and causes an increase in working time. In addition to this, opening and closing the reaction vessel containing the virus-containing sample poses a 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, which does not have a lid opening / closing step, has more significance than simplification of the work.


　前記混合液に添加される１ステップＲＴ－ＰＣＲ溶液は、（ｉ）逆転写酵素およびＤＮＡポリメラーゼを含むか、（ｉｉ）逆転写酵素活性を併せ持つＤＮＡポリメラーゼであるを含む。（ｉｉ）逆転写活性を有するＤＮＡポリメラーゼとしては、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。一つの好ましい実施形態では、二種の酵素、例えば、逆転写酵素とＤＮＡポリメラーゼの少なくとも２種類の酵素を使用することが好適である。
The one-step RT-PCR solution added to the mixture comprises (i) a reverse transcriptase and a DNA polymerase, or (ii) a DNA polymerase having both reverse transcriptase activity. (Ii) As the DNA polymerase having reverse transcription activity, it is preferable to use Tth DNA polymerase, Taq DNA polymerase, or the like. In one preferred embodiment, it is preferred to use two enzymes, for example, 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（ＲＴ）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、モロニーマウス白血病ウイルス（ＭＭＲＶ）由来逆転写酵素（ＭＭＬＶ－ＲＴ）、トリ骨髄芽球症ウイルス（ＡＭＶ）由来逆転写酵素（ＡＭＶ－ＲＴ）、またはそれらの変異体が挙げられる。
The origin of the reverse transcriptase (RT) 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 (Moloney Murine Leukemia Virus) -RT, AMV-RT (Avian Myeloblastosis Virus). ), HIV-RT, RAV2-RT, EIAV-RT, Carboxydothermus hydrogenoformam DNA polymerase) and variants thereof. Particularly preferred examples include reverse transcriptase derived from Moloney murine leukemia virus (MMRV) (MMLV-RT), reverse transcriptase derived from avian myeloblastosis virus (AMV) (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 mutants 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 DNA polymerase during the reverse transcription reaction can be achieved by introducing it into a DNA polymerase of a thermally 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、逆転写酵素またはＤＮＡポリメラーゼの変異体とは、その由来である野生型逆転写酵素または野生型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、野生型逆転写酵素または野生型ＤＮＡポリメラーゼと同様にＤＮＡを増幅する活性及び／又は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逆転写酵素または野生型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、野生型逆転写酵素または野生型ＤＮＡポリメラーゼと同様にＲＮＡをｃＤＮＡに変換する活性及び／又は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。
In the present specification, the variant of reverse transcriptase or DNA polymerase refers to, for example, 85% or more, preferably 90% or more, based on the amino acid sequence of wild-type reverse transcriptase or wild-type DNA polymerase from which it is derived. It has a sequence identity of preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more, and has an activity of amplifying DNA in the same manner as wild reverse transcriptase or wild DNA polymerase and / /. Alternatively, it refers to an enzyme having an 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 an analysis tool that is commercially available or available through the telecommunications line (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 reverse transcriptase or wild-type DNA polymerase from which it is derived (hereinafter, , Collectively referred to as "mutation"), and amplifies the activity of converting RNA into DNA and / or DNA in the same manner as wild reverse transcriptase or wild DNA polymerase. It may have activity. Here, 1 or several means, 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、逆転写酵素や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。
In addition to reverse transcriptase and DNA polymerase, the 1-step RT-PCR reaction solution used in the present invention contains a buffer, a magnesium salt or a manganese salt as an appropriate salt, a deoxynucleotide triphosphate, and a 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１ステップＲＴ－ＰＣＲ反応液中に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 1-step RT-PCR reaction solution 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, exhibits properties like a surfactant, and is thought 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の添加量は、本発明の効果を奏する限り限定されないが、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 amount of bovine serum albumin added to the one-step RT-PCR reaction solution is not limited as long as the effects of the present invention are exhibited, but is preferably at least 0.5 mg / ml or more, and 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 1-step RT-PCR reaction solution is derived from the skin, bones, 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 that derived from fish is preferable to that derived from cattle or pig because the jelly strength is low and the reaction solution is easy to handle.


　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、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, glycerol, ethylene glycol, propylene glycol, tri. Methylene glycol, formamide, acetamide, betaine, ectine, trehalose, dextran, polyvinylpyrrolidone (PVP), tetramethylammonium chloride (TMA), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), polyethylene glycol, triton Examples thereof include, but are not limited to, X-100 (TritonX-100), Triton X-114 (TritonX-114), Tween 20 (Tween 20), Nonidet P40, Briji58 and the like. Ethylene glycol-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から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。前記記載のプライマー配列では、配列番号１と２および配列番号４と５によりＳＡＲＳ－ｎＣＯＶ－２のヌクレオカプシドタンパク質（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、ヌクレオカプシド（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－ｄｅｐｅｎｄｅｎｔＲＮＡ ｐｏｌｙｍｅｒａｓｅ（ＲｄＲｐ）領域、ＯｐｅｎＲｅａｄｉｎｇ 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。
The primer pair used in the present invention is a pair of primers corresponding to the detection 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 are used. 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, degenerate primers may be included if the target nucleic acid consists of subtypes. When detecting coronavirus (SARS-nCOV-2), which is one of the enveloped RNA viruses, in the present invention, as an example of a primer pair, "Pathogen Detection Manual 2019-nCoV" published by the National Institute of Infectious Diseases. (SEQ ID NOS: 1 to 6) can be mentioned and can be preferably used in the present invention, but the present invention is not limited to this. In the primer sequences described above, the nucleocapsid protein (N) region of SARS-nCOV-2 is detected by SEQ ID NOs: 1 and 2 and SEQ ID NOs: 4 and 5. In the detection of coronaviruses such as SARS-nCOV-2, nucleocapsid (N) region, envelope protein (E) region, spike protein (S) region, RNA-dependentRNA polymerase (RdRp) region, open reading frame (ORF) Genes such as regions can be detected, but the detection is not limited to this. As for 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 that further comprises 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 effort 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。コロナウイルス検出用のプローブの塩基配列としては、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３および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). Examples of the base sequence of the probe for coronavirus detection include the sequences (SEQ ID NOs: 3 and 6) described in the "Pathogen Detection Manual 2019-nCoV" published by the National Institute of Infectious Diseases, which is also suitable for the present invention. It can be used for, but it 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, and ORF region can be detected, but the detection is not 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 further 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、アルカリ性溶液を含む前処理液、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、少なくともアルカリ性溶液を含む試薬、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。また、アルカリ性溶液を含む試薬は、界面活性剤を含まない試薬であってもよい。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。
Another aspect of the present invention is a kit for testing viral RNA in a sample, which is a pretreatment solution containing an alkaline solution, and reverse transcriptase and DNA polymerase (or DNA polymerase having reverse transcription activity), one step. A kit for testing enveloped RNA virus, which comprises an RT-PCR reaction solution. The virus testing kit of the present invention contains at least a reagent containing an alkaline solution, reverse transcriptase, DNA polymerase, and a one-step RT-PCR reaction solution. Further, the reagent containing an alkaline solution may be a reagent containing no surfactant. 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.


　本発明の前記ウイルス検査用キットは、核酸の分離精製処理を行っていない試料からエンベロープを持つウイルスの有無を検査するために用いることができる。本発明の前記ウイルスキットは、前述のような本発明のウイルス検査方法において好適に用いることができることから、例えば、本発明の前記ウイルス検査用キットは、核酸の分離精製処理を行っていない試料とｐＨ８以上のアルカリ性溶液との混合液の調製から１分以上経過した後に、１ステップＲＴ－ＰＣＲ反応液を添加し、反応容器を密閉後、１ステップＲＴ－ＰＣＲ反応を実施するようにして用いられ得る。このような本発明のウイルス検査用キットは、任意のエンベローブを持つウイルスの検査に使用できるが、好ましくは、コロナウイルスの検査用キットとして用いられ、より好ましくは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の検査用キットとして用いられ得る。
The virus test kit of the present invention can be used to test for the presence or absence of enveloped virus in a sample that has not been subjected to the separation and purification treatment of nucleic acid. Since the virus kit of the present invention can be suitably used in the virus test method of the present invention as described above, for example, the virus test kit of the present invention is a sample that has not been subjected to nucleic acid separation and purification treatment. After 1 minute or more has passed from the preparation of the mixed solution with the alkaline solution having a pH of 8 or more, the 1-step RT-PCR reaction solution is added, the reaction vessel is sealed, and then the 1-step RT-PCR reaction is carried out. obtain. Such a virus test kit of the present invention can be used for testing a virus having an arbitrary envelope, but is preferably used as a coronavirus test kit, and more preferably SARS (Severe Acute Respiratory Syndrome) corona. It can be used as a test kit for the virus, MERS (Middle East Respiratory Syndrome) coronavirus, SARS-nCOV-2.


　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．ｐＨを変更した条件での陰性糞便懸濁液の前処理検討

（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において、反応液中のコロナウイルスＲＮＡを検出した。プライマーおよびプローブ以外に関しては、（ノロウイルス検出キットＧ１／Ｇ２－高速プローブ検出Ｑｕｉｃｋ Ｓｔｅｐ－（東洋紡）添付品）を用いた。また、プライマーおよびプローブは「病原体検出マニュアル２０１９－ｎＣｏＶ」に記載の配列を使用し、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（４７μＬ）

　反応液：３０μＬ

　酵素液：５μＬ

　１０μＭ Ｎ＿Ｓａｒｂｅｃｏ＿Ｆ１（プライマー、配列番号１）：３μＬ

　１０μＭ Ｎ＿Ｓａｒｂｅｃｏ＿Ｒ１（プライマー、配列番号２）：４μＬ

　１０μＭ Ｎ＿Ｓａｒｂｅｃｏ＿Ｐ１（プローブ、配列番号３）：１μＬ

　ＲＮＡｓｅ ｆｒｅｅｗａｔｅｒ：４μＬ

（２）前処理液の調製

以下の組成の前処理液を調製した。

条件１

１０ｍＭ　酢酸－酢酸ナトリウム緩衝液（ｐＨ５．０）

条件２

１０ｍＭ　トリス－塩酸緩衝液（ｐＨ７．０）

条件３

１０ｍＭ　トリス－塩酸緩衝液（ｐＨ８．０）

条件４

１０ｍＭ　トリス－塩酸緩衝液（ｐＨ９．０）

条件５

１０ｍＭ　グリシン－水酸化ナトリウム緩衝液（ｐＨ９．５）

（３）試料の前処理

　（２）にて調製した各前処理液 １μＬに、陰性糞便懸濁液１μＬを混合し、室温（２５℃）３分間にて静置する条件、またはただちに次工程に移る条件の２条件にて検討を行った。

（４）前処理済各検体と鋳型ＲＮＡの添加

　前処理済の各検体２μＬに、コロナウイルスＮ１ ＲＮＡ（日本遺伝子研究所：ＳＡＲＳ－ｎＣＯＶ－２ ＲＮＡ）を最終濃度５０コピー／反応となるように１μＬを混合し、（１）にて調製したＲＴ－ＰＣＲ反応液４７μＬを添加して、５０μＬ反応系としてＲＴ－ＰＣＲを実施した。なお、ここまでの作業はすべて同一容器内において実施し、ＲＴ－ＰＣＲ反応液を添加後に容器を密閉してから一度もフタを開閉することなく１ステップＲＴ－ＰＣＲ反応を行った。

（５）ＲＴ－ＰＣＲ反応条件

　これをＲｏｃｈｅ製Ｌｉｇｈｔ Ｃｙｃｌｅｒ ９６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℃　１０分（逆転写条件）

　９５℃　１分   （熱変性）

　９８℃　１５秒－５５℃ １５秒－６０℃　４５秒 ５０サイクル（ＰＣＲ－蛍光読み取り）

（６）結果

　測定結果は、Ｒｏｃｈｅ製Ｌｉｇｈｔ Ｃｙｃｌｅｒ ９６解析ソフトウェアにてＣｔ値を算出し、Ｃｔ値が５０未満で算出された条件を陽性とした。結果、前処理液のｐＨ条件がｐＨ８．０以上にて、静置時間を３分間おくことで検出可能になることを確認した。本試験結果から、アルカリ性溶液と混合することでＲＴ－ＰＣＲ反応を阻害する夾雑物を多量に含む試料をそのまま用いて、１ステップＲＴ－ＰＣＲ反応によって十分な感度でＳＡＲＳ－ｎＣＯＶ－２を検出できることが示された。

Figure JPOXMLDOC01-appb-T000001

Test example 1. Examination of pretreatment of negative fecal suspension under the condition of changing pH

(1) Preparation of reaction solution

Using the reaction solution having the composition shown below as the basic composition, coronavirus RNA in the reaction solution was detected by 1-step RT-PCR. Other than the primers and probes, (Norovirus detection kit G1 / G2-fast probe detection Quick Step- (Toyobo) attachment) was used. In addition, the primers and probes used were the sequences described in the "Pathogen Detection Manual 2019-nCoV", and the probes were modified with FAM as the fluorescent label and BHQ1 (Black hole quencher) as the quenching group.

RT-PCR reaction solution (47 μL)

Reaction solution: 30 μL

Enzyme solution: 5 μL

10 μM N_Sarbeco_F1 (primer, SEQ ID NO: 1): 3 μL

10 μM N_Sarbeco_R1 (primer, SEQ ID NO: 2): 4 μL

10 μM N_Sarbeco_P1 (probe, SEQ ID NO: 3): 1 μL

RNAse freewater: 4 μL

(2) Preparation of pretreatment liquid

A pretreatment solution having the following composition was prepared.

Condition 1

10 mM acetate-sodium acetate buffer (pH 5.0)

Condition 2

10 mM Tris-hydrochloric acid buffer (pH 7.0)

Condition 3

10 mM Tris-hydrochloric acid buffer (pH 8.0)

Condition 4

10 mM Tris-hydrochloric acid buffer (pH 9.0)

Condition 5

10 mM glycine-sodium hydroxide buffer (pH 9.5)

(3) Sample pretreatment

1 μL of each pretreatment solution prepared in (2) is mixed with 1 μL of negative fecal suspension and allowed to stand at room temperature (25 ° C.) for 3 minutes, or immediately moved to the next step. Study was carried out.

(4) Addition of each pretreated sample and template RNA

2 μL of each pretreated sample was mixed with 1 μL of coronavirus N1 RNA (Japan Genetic Research Institute: SARS-nCOV-2 RNA) so as to have a final concentration of 50 copies / reaction, and RT prepared in (1). -PCR reaction solution 47 μL was added, and RT-PCR was performed as a 50 μL reaction system. All the operations up to this point were carried out in the same container, and after adding the RT-PCR reaction solution, the container was sealed and then the one-step RT-PCR reaction was carried out without opening or closing the lid.

(5) RT-PCR reaction conditions

This was subjected to a real-time PCR reaction using Roche's Light Cycler 96 in the following temperature cycle.

42 ° C for 10 minutes (reverse transfer conditions)

95 ° C for 1 minute (heat denaturation)

98 ° C 15 seconds-55 ° C 15 seconds-60 ° C 45 seconds 50 cycles (PCR-fluorescence reading)

(6) Result

As for the measurement result, the Ct value was calculated by the Light Cycler 96 analysis software manufactured by Roche, and the condition calculated when the Ct value was less than 50 was regarded as positive. As a result, it was confirmed that the pH condition of the pretreatment liquid was pH 8.0 or higher, and the detection became possible by leaving the standing time for 3 minutes. From the results of this test, SARS-nCOV-2 can be detected with sufficient sensitivity by a one-step RT-PCR reaction using a sample containing a large amount of impurities that inhibit the RT-PCR reaction when mixed with an alkaline solution. It has been shown.

Figure JPOXMLDOC01-appb-T000001


試験例２．アルカリ性溶液との混合後の経過時間の検討

（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において、反応液中のコロナウイルスＲＮＡを検出した。プライマーおよびプローブ以外に関しては、（ノロウイルス検出キットＧ１／Ｇ２－高速プローブ検出Ｑｕｉｃｋ Ｓｔｅｐ－（東洋紡）添付品）を用いた。また、プライマーおよびプローブは「病原体検出マニュアル２０１９－ｎＣｏＶ」に記載の配列を使用し、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（４７μＬ）

　反応液：３０μＬ

　酵素液：５μＬ

　１０μＭ Ｎ＿Ｓａｒｂｅｃｏ＿Ｆ１（プライマー、配列番号１）：３μＬ

　１０μＭ Ｎ＿Ｓａｒｂｅｃｏ＿Ｒ１（プライマー、配列番号２）：４μＬ

　１０μＭ Ｎ＿Ｓａｒｂｅｃｏ＿Ｐ１（プローブ、配列番号３）：１μＬ

　ＲＮＡｓｅ ｆｒｅｅｗａｔｅｒ：４μＬ

（２）検体の前処理と鋳型ＲＮＡの添加

前処理液（１０ｍＭ　トリス－塩酸緩衝液（ｐＨ　８．０））１μＬに、陰性糞便懸濁液１μＬを混合し、混合液の状態で室温（２５℃）にて０～３分間静置した。前処理済の各混合液にコロナウイルスＮ１ ＲＮＡ（日本遺伝子研究所：ＳＡＲＳ－ｎＣＯＶ－２ ＲＮＡ）を最終濃度５０コピー／反応となるように１μＬを混合し、（１）にて調製したＲＴ－ＰＣＲ反応液４７μＬを添加して、５０μＬ反応系としてＲＴ－ＰＣＲを実施した。なお、ここまでの作業はすべて同一容器内において実施し、ＲＴ－ＰＣＲ反応液を添加後に容器を密閉してから一度もフタを開閉することなく１ステップＲＴ－ＰＣＲ反応を行った。

（３）ＲＴ－ＰＣＲ反応条件

　これをＲｏｃｈｅ製Ｌｉｇｈｔ Ｃｙｃｌｅｒ ９６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℃　１０分（逆転写条件）

　９５℃　１分   （熱変性）

　９８℃　１５秒－５５℃ １５秒－６０℃　４５秒 ５０サイクル（ＰＣＲ－蛍光読み取り）

（４）結果

　測定結果は、Ｒｏｃｈｅ製Ｌｉｇｈｔ Ｃｙｃｌｅｒ ９６解析ソフトウェアにてＣｔ値を算出し、Ｃｔ値が５０未満で算出された条件を陽性とした。結果、前処理液との混合液の状態で少なくとも１分間経過することでＳＡＲＳ－ｎＣＯＶ－２ＲＮＡが検出可能になることを確認した。

Figure JPOXMLDOC01-appb-T000002

Test example 2. Examination of elapsed time after mixing with alkaline solution

(1) Preparation of reaction solution

Using the reaction solution having the composition shown below as the basic composition, coronavirus RNA in the reaction solution was detected by 1-step RT-PCR. Other than the primers and probes, (Norovirus detection kit G1 / G2-fast probe detection Quick Step- (Toyobo) attachment) was used. In addition, the primers and probes used were the sequences described in the "Pathogen Detection Manual 2019-nCoV", and the probes were modified with FAM as the fluorescent label and BHQ1 (Black hole quencher) as the quenching group.

RT-PCR reaction solution (47 μL)

Reaction solution: 30 μL

Enzyme solution: 5 μL

10 μM N_Sarbeco_F1 (primer, SEQ ID NO: 1): 3 μL

10 μM N_Sarbeco_R1 (primer, SEQ ID NO: 2): 4 μL

10 μM N_Sarbeco_P1 (probe, SEQ ID NO: 3): 1 μL

RNAse freewater: 4 μL

(2) Specimen pretreatment and addition of template RNA

1 μL of the negative fecal suspension was mixed with 1 μL of the pretreatment solution (10 mM Tris-hydrochloric acid buffer (pH 8.0)), and the mixture was allowed to stand at room temperature (25 ° C.) for 0 to 3 minutes. 1 μL of coronavirus N1 RNA (Japan Genetic Research Institute: SARS-nCOV-2 RNA) was mixed with each pretreated mixture so as to have a final concentration of 50 copies / reaction, and RT- prepared in (1). 47 μL of PCR reaction solution was added, and RT-PCR was performed as a 50 μL reaction system. All the operations up to this point were carried out in the same container, and after adding the RT-PCR reaction solution, the container was sealed and then the one-step RT-PCR reaction was carried out without opening or closing the lid.

(3) RT-PCR reaction conditions

This was subjected to a real-time PCR reaction using Roche's Light Cycler 96 in the following temperature cycle.

42 ° C for 10 minutes (reverse transfer conditions)

95 ° C for 1 minute (heat denaturation)

98 ° C 15 seconds-55 ° C 15 seconds-60 ° C 45 seconds 50 cycles (PCR-fluorescence reading)

(4) Result

As for the measurement result, the Ct value was calculated by the Light Cycler 96 analysis software manufactured by Roche, and the condition calculated when the Ct value was less than 50 was regarded as positive. As a result, it was confirmed that SARS-nCOV-2RNA became detectable after at least 1 minute had passed in the state of a mixed solution with the pretreatment solution.

Figure JPOXMLDOC01-appb-T000002


試験例３．その他の生体由来検体存在下での検出検討

（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において、反応液中のコロナウイルスＲＮＡを検出した。プライマーおよびプローブ以外に関しては、（ノロウイルス検出キットＧ１／Ｇ２－高速プローブ検出Ｑｕｉｃｋ Ｓｔｅｐ－（東洋紡）添付品）を用いた。また、プライマーおよびプローブは「病原体検出マニュアル２０１９－ｎＣｏＶ 」に記載の配列を使用し、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（４７μＬ）

条件１．Ｎ領域１

　反応液：３０μＬ

　酵素液：５μＬ

　１０μＭ Ｎ＿Ｓａｒｂｅｃｏ＿Ｆ１（プライマー、配列番号１）：３μＬ

　１０μＭ Ｎ＿Ｓａｒｂｅｃｏ＿Ｒ１（プライマー、配列番号２）：４μＬ

　１０μＭ Ｎ＿Ｓａｒｂｅｃｏ＿Ｐ１（プローブ、配列番号３）：１μＬ

　ＲＮＡｓｅ ｆｒｅｅｗａｔｅｒ：４μＬ

（２）各検体の前処理

　前処理液（１０ｍＭトリス－塩酸緩衝液（ｐＨ８．０））１μＬに、咽頭ぬぐい液、鼻腔ぬぐい液をそれぞれ１μＬを添加して混合した。混合後、室温にて１分間静置した。咽頭ぬぐい液、鼻腔ぬぐい液は、該当箇所をスワブに採取後ＵＴＭウイルス輸送用培地（コパン）に懸濁したものを利用した。

（３）前処理済各検体と鋳型ＲＮＡの添加

　前処理済の各検体２μＬに、コロナウイルスＮ１ ＲＮＡ（日本遺伝子研究所：ＳＡＲＳ－ｎＣＯＶ－２ ＲＮＡ）を最終濃度５０コピー／反応となるように１μＬを混合し、（１）にて調製したＲＴ－ＰＣＲ反応液４７μＬを添加して、５０μＬ反応系としてＲＴ－ＰＣＲを実施した。

（４）ＲＴ－ＰＣＲ反応条件

　これをＲｏｃｈｅ製Ｌｉｇｈｔ Ｃｙｃｌｅｒ ９６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℃　１０分（逆転写条件）

　９５℃　１分   （熱変性）

　９８℃　１５秒－５５℃ １５秒－６０℃　４５秒 ５０サイクル（ＰＣＲ－蛍光読み取り）

（５）結果

　測定結果は、Ｒｏｃｈｅ製Ｌｉｇｈｔ Ｃｙｃｌｅｒ ９６解析ソフトウェアにてＣｔ値を算出し、Ｃｔ値が５０未満で算出された条件を陽性とした。結果、全ての条件において、ＳＡＲＳ－ｎＣＯＶ－２ＲＮＡが検出可能であることを確認した。

Figure JPOXMLDOC01-appb-T000003

Test example 3. Examination of detection in the presence of other biological specimens

(1) Preparation of reaction solution

Using the reaction solution having the composition shown below as the basic composition, coronavirus RNA in the reaction solution was detected by 1-step RT-PCR. Other than the primers and probes, (Norovirus detection kit G1 / G2-fast probe detection Quick Step- (Toyobo) attachment) was used. In addition, the primers and probes used were the sequences described in the "Pathogen Detection Manual 2019-nCoV", and the probes were modified with FAM as the fluorescent label and BHQ1 (Black hole quencher) as the quenching group.

RT-PCR reaction solution (47 μL)

Condition 1. N area 1

Reaction solution: 30 μL

Enzyme solution: 5 μL

10 μM N_Sarbeco_F1 (primer, SEQ ID NO: 1): 3 μL

10 μM N_Sarbeco_R1 (primer, SEQ ID NO: 2): 4 μL

10 μM N_Sarbeco_P1 (probe, SEQ ID NO: 3): 1 μL

RNAse freewater: 4 μL

(2) Pretreatment of each sample

To 1 μL of the pretreatment solution (10 mM Tris-hydrochloric acid buffer (pH 8.0)), 1 μL of each of the pharyngeal swab and the nasal swab was added and mixed. After mixing, it was allowed to stand at room temperature for 1 minute. As the pharyngeal swab and the nasal swab, the relevant parts were collected in a swab and then suspended in a UTM virus transport medium (Copan).

(3) Addition of each pretreated sample and template RNA

2 μL of each pretreated sample was mixed with 1 μL of coronavirus N1 RNA (Japan Genetic Research Institute: SARS-nCOV-2 RNA) so as to have a final concentration of 50 copies / reaction, and RT prepared in (1). -PCR reaction solution 47 μL was added, and RT-PCR was performed as a 50 μL reaction system.

(4) RT-PCR reaction conditions

This is performed using Roche's Light Cycler 96 in the following temperature cycle.

A real-time PCR reaction was performed.

42 ° C for 10 minutes (reverse transfer conditions)

95 ° C for 1 minute (heat denaturation)

98 ° C 15 seconds-55 ° C 15 seconds-60 ° C 45 seconds 50 cycles (PCR-fluorescence reading)

(5) Result

As for the measurement result, the Ct value was calculated by the Light Cycler 96 analysis software manufactured by Roche, and the condition calculated when the Ct value was less than 50 was regarded as positive. As a result, it was confirmed that SARS-nCOV-2RNA was detectable under all conditions.

Figure JPOXMLDOC01-appb-T000003


試験例４．ｐＨを変更した条件での不活化ウイルスに対する前処理効果の検討

（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において、反応液中の不活化されたＳＡＲＳ－ＣｏＶ－２ウイルスを検出した。プライマーおよびプローブ以外に関しては、（ノロウイルス検出キットＧ１／Ｇ２－高速プローブ検出 Ｑｕｉｃｋ Ｓｔｅｐ－（東洋紡）添付品）を用いた。また、プライマーおよびプローブは「病原体検出マニュアル２０１９－ｎＣｏＶ」に記載の配列を使用し、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（４８μＬ）

　反応液：３０μＬ

　酵素液：５μＬ

　１０μＭ ＮＩＩＤ＿２０１９－ｎＣＯＶ＿Ｎ＿Ｆ２（プライマー、配列番号４）：２．５μＬ

　１０μＭ ＮＩＩＤ＿２０１９－ｎＣＯＶ＿Ｎ＿Ｒ２（プライマー、配列番号５）：３．５μＬ

　１０μＭ ＮＩＩＤ＿２０１９－ｎＣＯＶ＿Ｎ＿Ｐ２（プローブ、配列番号６）：１μＬ

　ＲＮＡｓｅ ｆｒｅｅ ｗａｔｅｒ：６μＬ

（２）前処理液の調製

以下の組成の前処理液を調製した。

条件１ 

１０ｍＭ　トリス－塩酸緩衝液（ｐＨ８．０）

条件２

１０ｍＭ　トリス－塩酸緩衝液（ｐＨ８．５）

条件３

１０ｍＭ　トリス－塩酸緩衝液（ｐＨ９．０）

条件４

１０ｍＭ　グリシン－水酸化ナトリウム緩衝液（ｐＨ９．５）

（３）不活化ウイルスの前処理

　各前処理液１μＬに、１０コピー／μＬとなるように調製した不活化ＳＡＲＳ－ｎＣＯＶ－２ウイルス（Ｉｎａｃｔｉｖａｔｅｄ　ＳＡＲＳ－ＣｏＶ－２（２０１９－ｎＣｏＶ／ＵＳＡ－ＷＡ１／２０２０）（ＡＴＣＣ）)１μＬを混合し、混合液の状態で室温（２５℃）にて１分間静置した。陰性対照は１μＬのＲＮＡｓｅ ｆｒｅｅ ｗａｔｅｒとした。（１）にて調製したＲＴ－ＰＣＲ反応液４８μＬを添加して、５０μＬ反応系としてＲＴ－ＰＣＲを実施した。なお、ここまでの作業はすべて同一容器内において実施し、ＲＴ－ＰＣＲ反応液を添加後に容器を密閉してから一度もフタを開閉することなく１ステップＲＴ－ＰＣＲ反応を行った。

（４）ＲＴ－ＰＣＲ反応条件

　これをＢｉｏ－Ｒａｄ製ＣＦＸ９６　ＤＥＥＰ　ＷＥＬＬ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℃　１０分（逆転写条件）

　９５℃　１分   （熱変性）

　９８℃　１５秒－５５℃ １５秒－６０℃　４５秒 ５０サイクル（ＰＣＲ－蛍光読み取り）

（５）結果

　測定結果は、Ｂｉｏ－Ｒａｄ製ＣＦＸ９６解析ソフトウェアにて、閾値＝１５０としてＣｔ値を算出し、Ｃｔ値が５０未満で算出された条件を陽性とした。結果、前処理液のｐＨが８以上であれば、ＳＡＲＳ－ｎＣＯＶ－２ウイルスの検出が可能となることを確認した。

Figure JPOXMLDOC01-appb-T000004

Test example 4. Examination of pretreatment effect on inactivated virus under the condition of changing pH

(1) Preparation of reaction solution

Using the reaction solution having the composition shown below as the basic composition, the inactivated SARS-CoV-2 virus in the reaction solution was detected by 1-step RT-PCR. Other than the primers and probes, (Norovirus detection kit G1 / G2-fast probe detection Quick Step- (Toyobo) attachment) was used. In addition, the primers and probes used were the sequences described in the "Pathogen Detection Manual 2019-nCoV", and the probes were modified with FAM as the fluorescent label and BHQ1 (Black hole quencher) as the quenching group.

RT-PCR reaction solution (48 μL)

Reaction solution: 30 μL

Enzyme solution: 5 μL

10 μM NIID_2019-nCOV_N_F2 (primer, SEQ ID NO: 4): 2.5 μL

10 μM NIID_2019-nCOV_N_R2 (primer, SEQ ID NO: 5): 3.5 μL

10 μM NIID_2019-nCOV_N_P2 (probe, SEQ ID NO: 6): 1 μL

RNAse free water: 6 μL

(2) Preparation of pretreatment liquid

A pretreatment solution having the following composition was prepared.

Condition 1

10 mM Tris-hydrochloric acid buffer (pH 8.0)

Condition 2

10 mM Tris-hydrochloric acid buffer (pH 8.5)

Condition 3

10 mM Tris-hydrochloric acid buffer (pH 9.0)

Condition 4

10 mM glycine-sodium hydroxide buffer (pH 9.5)

(3) Pretreatment of inactivated virus

1 μL of each pretreatment solution is mixed with 1 μL of inactivated SARS-nCOV-2 virus (Inactivated SARS-CoV-2 (2019-nCoV / USA-WA1 / 2020) (ATCC)) prepared to be 10 copies / μL. Then, the mixture was allowed to stand at room temperature (25 ° C.) for 1 minute. The negative control was 1 μL of RNAse free water. 48 μL of the RT-PCR reaction solution prepared in (1) was added, and RT-PCR was carried out as a 50 μL reaction system. All the operations up to this point were carried out in the same container, and after adding the RT-PCR reaction solution, the container was sealed and then the one-step RT-PCR reaction was carried out without opening or closing the lid.

(4) RT-PCR reaction conditions

This was subjected to a real-time PCR reaction using CFX96 DEEP WELL manufactured by Bio-Rad in the following temperature cycle.

42 ° C for 10 minutes (reverse transfer conditions)

95 ° C for 1 minute (heat denaturation)

98 ° C 15 seconds-55 ° C 15 seconds-60 ° C 45 seconds 50 cycles (PCR-fluorescence reading)

(5) Result

As for the measurement result, the Ct value was calculated with the CFX96 analysis software manufactured by Bio-Rad with the threshold value = 150, and the condition calculated when the Ct value was less than 50 was regarded as positive. As a result, it was confirmed that SARS-nCOV-2 virus can be detected when the pH of the pretreatment solution is 8 or more.

Figure JPOXMLDOC01-appb-T000004


試験例５．その他の生体由来検体存在下での検出検討

（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において、反応液中の不活化されたＳＡＲＳ－ＣｏＶ－２ウイルスを検出した。プライマーおよびプローブ以外に関しては、（ノロウイルス検出キットＧ１／Ｇ２－高速プローブ検出 Ｑｕｉｃｋ Ｓｔｅｐ－（東洋紡）添付品）を用いた。また、プライマーおよびプローブは「病原体検出マニュアル２０１９－ｎＣｏＶ 」に記載の配列を使用し、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（４６μＬ）

　反応液：３０μＬ

　酵素液：５μＬ

　１０μＭ ＮＩＩＤ＿２０１９－ｎＣＯＶ＿Ｎ＿Ｆ２（プライマー、配列番号４）：２．５μＬ

　１０μＭ ＮＩＩＤ＿２０１９－ｎＣＯＶ＿Ｎ＿Ｒ２（プライマー、配列番号５）：３．５μＬ

　１０μＭ ＮＩＩＤ＿２０１９－ｎＣＯＶ＿Ｎ＿Ｐ２（プローブ、配列番号６）：１μＬ

　ＲＮＡｓｅ ｆｒｅｅ ｗａｔｅｒ：４μＬ

（２）各検体の前処理

　前処理液（１０ｍＭ　グリシン－水酸化ナトリウム緩衝液（ｐＨ９．５））２μＬに、２５コピー／μＬとなるように調製した不活化ＳＡＲＳ－ｎＣＯＶ－２ウイルス（Ｉｎａｃｔｉｖａｔｅｄ　ＳＡＲＳ－ＣｏＶ－２　（２０１９－ｎＣｏＶ／ＵＳＡ－ＷＡ１／２０２０）（ＡＴＣＣ）)１μＬと、唾液、咽頭ぬぐい液、鼻腔ぬぐい液をそれぞれ１μＬ添加して混合した。混合後、室温にて１分間静置した。前処理済の各検体４μＬに、（１）にて調製したＲＴ－ＰＣＲ反応液４６μＬを添加して、５０μＬ反応系としてＲＴ－ＰＣＲを実施した。

　なお、ここまでの作業はすべて同一容器内において実施し、ＲＴ－ＰＣＲ反応液を添加後に容器を密閉してから一度もフタを開閉することなく１ステップＲＴ－ＰＣＲ反応を行った。なお、各検体は以下のようにして調製した。唾液はプラスチック容器に直接採取後、唾液１００μＬとＰＢＳ１００μＬを等量混合し、その後、遠心分離（１５０００ｒｐｍ、５分間）して上清を回収し、検体とした。咽頭ぬぐい液、鼻腔ぬぐい液は、該当箇所からスワブを用いて採取し、ＵＴＭウイルス輸送用培地（コパン）に懸濁したものを利用した。前処理液の陰性対照は２μＬのＲＮＡｓｅ ｆｒｅｅ ｗａｔｅｒとした。

（３）ＲＴ－ＰＣＲ反応条件

　これをＢｉｏ－Ｒａｄ製ＣＦＸ９６　ＤＥＥＰ　ＷＥＬＬ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℃　１０分（逆転写条件）

　９５℃　１分   （熱変性）

　９８℃　１５秒－５５℃ １５秒－６０℃　４５秒 ５０サイクル（ＰＣＲ－蛍光読み取り）

（４）結果

　測定結果は、Ｂｉｏ－Ｒａｄ製ＣＦＸ９６解析ソフトウェアにて、閾値＝１５０としてＣｔ値を算出し、Ｃｔ値が５０未満で算出された条件を陽性とした。結果、ｐＨ９．５にて前処理した全ての条件において、生体試料存在下におけるＳＡＲＳ－ｎＣＯＶ－２ウイルスが検出可能となることを確認した。

Figure JPOXMLDOC01-appb-T000005

Test example 5. Examination of detection in the presence of other biological specimens

(1) Preparation of reaction solution

Using the reaction solution having the composition shown below as the basic composition, the inactivated SARS-CoV-2 virus in the reaction solution was detected by 1-step RT-PCR. Other than the primers and probes, (Norovirus detection kit G1 / G2-fast probe detection Quick Step- (Toyobo) attachment) was used. In addition, the primers and probes used were the sequences described in the "Pathogen Detection Manual 2019-nCoV", and the probes were modified with FAM as the fluorescent label and BHQ1 (Black hole quencher) as the quenching group.

RT-PCR reaction solution (46 μL)

Reaction solution: 30 μL

Enzyme solution: 5 μL

10 μM NIID_2019-nCOV_N_F2 (primer, SEQ ID NO: 4): 2.5 μL

10 μM NIID_2019-nCOV_N_R2 (primer, SEQ ID NO: 5): 3.5 μL

10 μM NIID_2019-nCOV_N_P2 (probe, SEQ ID NO: 6): 1 μL

RNAse free water: 4 μL

(2) Pretreatment of each sample

Inactivated SARS-nCOV-2 virus (Inactivated SARS-CoV-2 (2019-nCoV)) prepared to 25 copies / μL in 2 μL of pretreatment solution (10 mM glycine-sodium hydroxide buffer (pH 9.5)). / USA-WA1 / 2020) (ATCC)) 1 μL and saliva, pharyngeal swab, and nasal swab were added and mixed by 1 μL each. After mixing, it was allowed to stand at room temperature for 1 minute. To 4 μL of each pretreated sample, 46 μL of the RT-PCR reaction solution prepared in (1) was added, and RT-PCR was carried out as a 50 μL reaction system.

All the operations up to this point were carried out in the same container, and after adding the RT-PCR reaction solution, the container was sealed and then the one-step RT-PCR reaction was carried out without opening or closing the lid. Each sample was prepared as follows. Saliva was collected directly in a plastic container, 100 μL of saliva and 100 μL of PBS were mixed in equal amounts, and then centrifuged (15000 rpm, 5 minutes) to collect the supernatant, which was used as a sample. The pharyngeal and nasal swabs were collected from the relevant sites using a swab and suspended in a UTM virus transport medium (Copan). The negative control of the pretreatment solution was 2 μL of RNAse free water.

(3) RT-PCR reaction conditions

This was subjected to a real-time PCR reaction using CFX96 DEEP WELL manufactured by Bio-Rad in the following temperature cycle.

42 ° C for 10 minutes (reverse transfer conditions)

95 ° C for 1 minute (heat denaturation)

98 ° C 15 seconds-55 ° C 15 seconds-60 ° C 45 seconds 50 cycles (PCR-fluorescence reading)

(4) Result

As for the measurement result, the Ct value was calculated with the CFX96 analysis software manufactured by Bio-Rad with the threshold value = 150, and the condition calculated when the Ct value was less than 50 was regarded as positive. As a result, it was confirmed that SARS-nCOV-2 virus can be detected in the presence of a biological sample under all conditions of pretreatment at pH 9.5.

Figure JPOXMLDOC01-appb-T000005


　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 (23)


　試料中のエンベロープを持つ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）核酸の分離精製処理を行っていない試料とｐＨ８以上のアルカリ性溶液とを混合する工程、

（２）前記混合液の調製から１分以上経過した後に、（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、

（３）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 testing an RNA virus having an envelope in a sample, which comprises the following steps:

(1) A step of mixing a sample that has not been separated and purified from nucleic acid with an alkaline solution having a pH of 8 or higher.

(2) 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 after 1 minute or more has passed from the preparation of the mixed solution.

(3) A step of carrying out a one-step RT-PCR reaction after sealing the reaction vessel.
　前記工程（１）～（３）が同一容器で行われることを特徴とする請求項１に記載の検査方法。
The inspection method according to claim 1, wherein the steps (1) to (3) are performed in the same container.
　工程（３）において反応容器を密閉後、一度もフタを開閉することなく１ステップＲＴ－ＰＣＲ反応を実施することを特徴とする請求項１又は２に記載の検査方法。
The test method according to claim 1 or 2, wherein the reaction vessel is sealed in the step (3), and then the one-step RT-PCR reaction is carried out without opening and closing the lid even once.
　アルカリ性溶液が、水酸化カリウム水溶液、水酸化ナトリウム水溶液、水酸化リチウム水溶液、水酸化マグネシウム水溶液、水酸化カルシウム水溶液、水酸化バリウム水溶液、炭酸カリウム水溶液、炭酸ナトリウム水溶液、炭酸マグネシウム水溶液、炭酸カルシウム水溶液、トリス緩衝液、グリシン緩衝液、リン酸緩衝液、ホウ酸緩衝液、又はグッド緩衝液である請求項１から３のいずれかに記載の検査方法。
The alkaline solution is potassium hydroxide aqueous solution, sodium hydroxide aqueous solution, lithium hydroxide aqueous solution, magnesium hydroxide aqueous solution, calcium hydroxide aqueous solution, barium hydroxide aqueous solution, potassium carbonate aqueous solution, sodium carbonate aqueous solution, magnesium carbonate aqueous solution, calcium carbonate aqueous solution, The test method according to any one of claims 1 to 3, which is a Tris buffer solution, a glycine buffer solution, a phosphate buffer solution, a borate buffer solution, or a Good buffer solution.
　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、及び培養上清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４のいずれかに記載の検査方法。
The sample is at least one selected from the group consisting of feces, pharyngeal swab, nasal swab, sputum, lung aspirate, cerebrospinal fluid, mouthwash, saliva, tears, cultured cells, and culture supernatant. The inspection method according to any one of claims 1 to 4.
　試料が、水、生理食塩水または緩衝液に予め懸濁された懸濁液である請求項１から５のいずれかに記載の検査方法。
The test method according to any one of claims 1 to 5, wherein the sample is a suspension pre-suspended in water, saline or buffer.
　試料が、懸濁液の遠心上清である請求項６に記載の検査方法。
The inspection method according to claim 6, wherein the sample is a centrifugal supernatant of a suspension.
　試料が、環境中の拭き取り検査試料を水、生理食塩水または緩衝液に予め懸濁し、かつ、該懸濁液を濃縮した試料である請求項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 sample in which a wipe test sample in an environment is previously suspended in water, physiological saline or a buffer solution, and the suspension is concentrated.
　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１から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 having an envelope is a coronavirus.
　コロナウイルスが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、又はＳＡＲＳ－ｎＣＯＶ－２コロナウイルスである請求項９に記載の検査方法。
The test method according to claim 9, wherein the coronavirus is SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, or SARS-nCOV-2 coronavirus.
　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少なくとも1種である請求項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 DNA polymerase is at least one selected from the group consisting of Taq, Tth and variants thereof.
　逆転写酵素が、モロニーマウス白血病ウイルス（ＭＭＲＶ）由来逆転写酵素、トリ骨髄芽球症ウイルス（ＡＭＶ）由来逆転写酵素、およびこれらの変異体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１１のいずれかに記載の検査方法。
Claim that the reverse transcriptase is at least one selected from the group consisting of Moloney murine leukemia virus (MMRV) -derived reverse transcriptase, avian myeloblastosis virus (AMV) -derived reverse transcriptase, and variants thereof. The inspection method according to any one of 1 to 11.
　工程（３）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つを含む請求項１から１２のいずれかに記載の検査方法。
The 1-step RT-PCR reaction solution in step (3) 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 12, which comprises at least one selected from the group consisting of albumin, glycerol, glycol and gelatin.
　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１３に記載の検査方法。
The test method according to claim 13, wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine.
　ｐＨ８以上のアルカリ性溶液、逆転写酵素、ＤＮＡポリメラーゼ、および１ステップＲＴ－ＰＣＲ反応液を含むことを特徴とするエンベロープを持つウイルスの検査用キット。
A kit for testing a virus having an envelope, which comprises an alkaline solution having a pH of 8 or higher, reverse transcriptase, DNA polymerase, and a 1-step RT-PCR reaction solution.
　核酸の分離精製処理を行っていない試料から、エンベロープを持つウイルスの有無を検査するために用いられる、請求項１５に記載のウイルスの検査用キット。
The virus testing kit according to claim 15, which is used for testing for the presence or absence of an enveloped virus from a sample that has not been subjected to a nucleic acid separation and purification treatment.
　核酸の分離精製処理を行っていない試料とｐＨ８以上のアルカリ性溶液との混合液の調製から１分以上経過した後に、１ステップＲＴ－ＰＣＲ反応液を添加し、反応容器を密閉後、１ステップＲＴ－ＰＣＲ反応を実施するようにして用いられる、請求項１６に記載のウイルスの検査用キット。
After 1 minute or more has passed from the preparation of the mixed solution of the sample not subjected to the separation and purification treatment of nucleic acid and the alkaline solution having a pH of 8 or more, the 1-step RT-PCR reaction solution is added, the reaction vessel is sealed, and then the 1-step RT. -The kit for testing for viruses according to claim 16, which is used to carry out a PCR reaction.
　アルカリ性溶液が、水酸化カリウム水溶液、水酸化ナトリウム水溶液、水酸化リチウム水溶液、水酸化マグネシウム水溶液、水酸化カルシウム水溶液、水酸化バリウム水溶液、炭酸カリウム水溶液、炭酸ナトリウム水溶液、炭酸マグネシウム水溶液、炭酸カルシウム水溶液、トリス緩衝液、グリシン緩衝液、リン酸緩衝液、ホウ酸緩衝液、又はグッド緩衝液である、請求項１５から１７のいずれかに記載のウイルスの検査用キット。
The alkaline solution is potassium hydroxide aqueous solution, sodium hydroxide aqueous solution, lithium hydroxide aqueous solution, magnesium hydroxide aqueous solution, calcium hydroxide aqueous solution, barium hydroxide aqueous solution, potassium carbonate aqueous solution, sodium carbonate aqueous solution, magnesium carbonate aqueous solution, calcium carbonate aqueous solution, The virus test kit according to any one of claims 15 to 17, which is a Tris buffer solution, a glycine buffer solution, a phosphate buffer solution, a borate buffer solution, or a Good buffer solution.
　１ステップＲＴ－ＰＣＲ反応液が、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つを含む請求項１５から１８のいずれかに記載のウイルスの検査用キット。
The virus according to any one of claims 15 to 18, wherein the one-step RT-PCR reaction solution contains at least one selected from the group consisting of betaine-like quaternary ammonium salt, bovine serum albumin, glycerol, glycol and gelatin. Inspection kit.
　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１５から１９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 any one of claims 15 to 19, further comprising a primer pair corresponding to the detection region of the RNA virus to be detected.
　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１５から２０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 any one of claims 15 to 20, further comprising a hybridization probe corresponding to the detection region of the RNA virus to be detected.
　エンべロープを持つＲＮＡウイルスがコロナウイルスである請求項１５から２１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 any one of claims 15 to 21, wherein the RNA virus having an envelope is a coronavirus.
　コロナウイルスが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、又はＳＡＲＳ－ｎＣＯＶ－２であることを特徴とする請求項２２に記載のウイルスの検査用キット。
The virus test kit according to claim 22, wherein the coronavirus is SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, or SARS-nCOV-2.
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