JP2023024808A - Methods for detecting rna viruses - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods for detecting RNA viruses in test samples.

SOLUTION: The invention relates to a method for detecting an RNA virus by reverse transcription-polymerase chain reaction (RT-PCR), and a kit for conducting the method. More specifically, the invention relates to a method for detecting an RNA virus and a kit for conducting the method, which method comprises mixing a test sample with a sample processing fluid containing a surfactant followed by addition of an RT-PCR reaction solution.

SELECTED DRAWING: None

COPYRIGHT: (C)2023,JPO&INPIT

Description

The present invention relates to a method for detecting RNA viruses by reverse transcription-polymerase chain reaction (RT-PCR) and a kit for carrying out the method. More specifically, the present invention relates to a method for detecting RNA viruses by mixing a sample with a sample treatment solution containing a surfactant and adding an RT-PCR reaction solution, and a kit for carrying out the method.

RNA viruses are viruses that have RNA as a genome, and include coronaviruses, human immunodeficiency virus, hepatitis C virus, Japanese encephalitis virus, dengue virus, etc. that have an envelope that is a membrane consisting of a lipid bilayer, and norovirus that does not have an envelope. , rotavirus, and rhinovirus, many of which are pathogenic.

Norovirus is a virus belonging to the human caliciviridae family, and has a single-stranded RNA of about 7000 bases in its genome. This virus is also called Small Round Structured Virus (SRSV) due to its morphological classification observed with an electron microscope, and has been called the genus Norwalk-like virus. is. Viruses belonging to Norovirus are classified into two genogroups, Genogroup (GI) and Genogroup II (GII), and are further classified into 14 and 17 or more genotypes, respectively.

Norovirus infection in humans causes acute gastrointestinal symptoms such as vomiting and diarrhea. Approximately half of the annual food poisoning patients in Japan are caused by norovirus, of which approximately 70% occur from November to February, and norovirus is known to cause winter-type gastroenteritis and food poisoning. Norovirus food poisoning is mainly caused by contamination of food through cooks. Norovirus is highly contagious and easily causes mass outbreaks such as large-scale food poisoning. The route of infection to humans is mainly oral infection. The feces or vomit of an infected person, items directly or indirectly contaminated with these, and foods such as oysters or other bivalves contaminated with norovirus are typical sources of infection. Therefore, it is important to identify norovirus-infected patients and contaminants caused by the virus in order to prevent the spread of virus infection.

Viral tests for detecting viral infection and contamination include immunoassays for detecting viral antigens and viral gene amplification methods (Patent Documents 1 to 3, Non-Patent Document 1). As a means for measuring norovirus with high sensitivity, there is a method of amplifying norovirus RNA by RT-PCR and measuring the amount of amplified product. For example, detection of norovirus by RT-PCR method and quantitative detection of norovirus by real-time PCR method are widely performed in accordance with the notification by the Inspection and Safety Division, Food Safety Department, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor and Welfare (Non-Patent Documents 2 and 3). ing.

RNA virus particles have a basic structure in which a core consisting of an RNA genome and proteins is enclosed in a protein shell called a capsid. Therefore, in order to detect viral RNA by gene amplification, it is necessary to extract RNA from virus particles. To detect norovirus in stool as a sample, for example, a stool sample is suspended in distilled water or physiological saline at a concentration of 5 to 10% (w/v), and a commercially available viral RNA extraction is performed from the centrifugal supernatant. RNA is extracted and purified using a kit (eg, QIAamp (registered trademark) Viral RNA Mini, QIAGEN) (Non-Patent Document 2). However, the detection process in which RT-PCR is performed after multistep RNA extraction and purification is complicated. For this reason, a simple detection method in which a fecal suspension is mixed with a sample treatment solution, heat-treated for a short time to remove the shell protein, release the RNA inside, and subject the released RNA directly to RT-PCR. has been proposed (Non-Patent Document 4). On the other hand, in order to heat-treat the mixture of the stool suspension and sample treatment solution, the reaction vessel is sealed with a lid to prevent bumping and evaporation of the mixture, the lid is removed after the heat treatment, and the RT-PCR reaction solution is added. It takes time to do it. In order to improve this point, a method has been proposed in which a sample is mixed with a chaotropic agent such as a guanidine salt to detect viruses by RT-PCR without heat treatment (Patent Document 4).

WO2002/029119 WO 2002/029120 JP 2004-301684 A JP 2017-209036 A

Kageyama T, et al. Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. J Clin Microbiol. 2003 Apr;41(4):1548-57. Ministry of Health, Labor and Welfare, Pharmaceutical and Food Safety Bureau, Food Safety Department, Inspection and Safety Division Food Safety Inspection No. 1105001 (November 5, 2003) Attachment "Norovirus Detection Method", final revision: Food Safety Inspection No. 0514004 (May 2007) 14th) Ministry of Health, Labor and Welfare, Pharmaceutical and Food Safety Bureau, Food Safety Department, Inspection and Safety Division, Food Safety Inspection No. 1105001 (November 5, 2003) Attachment "Norovirus Detection Method", Final revision: Food Safety Inspection No. 1022 No. 1 (2013) October 22) Nishimura N, et al. Detection of noroviruses in fecal specimens by direct RT-PCR without RNA purification. J Virol Methods. 2010 Feb;163(2):282-286.

An object of the present invention is to provide a simple method for detecting RNA viruses. Specifically, using one or more surfactants, RNA extraction from RNA virus particles such as norovirus is performed without heat treatment, and the subsequent RT-PCR reaction of the released RNA is used to easily detect viruses. to provide a method of doing so.

The object of the present invention is achieved by the following inventions.
[1]
A method for detecting an RNA virus in a specimen, comprising:
(1) mixing a specimen with a specimen treatment liquid containing one or more surfactants to form a suspension;
(2) extracting the centrifugal supernatant from the suspension produced in step (1) by centrifugation;
(3) mixing the centrifugal supernatant extracted in step (2) with a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase to perform RT-PCR;
(4) detecting the RT-PCR product;
method including.
[2]
The method of [1], wherein the RNA virus is selected from the group consisting of norovirus, rotavirus, rhinovirus, coronavirus, human immunodeficiency virus, hepatitis C virus, Japanese encephalitis virus and dengue virus.
[3]
The method of [1], wherein the RNA virus is a norovirus.
[4]
The method of [3], wherein the Norovirus genotype is Genogroup I (GI) or Genogroup II (GII).
[5]
The method according to any one of [1] to [4], wherein the specimen is derived from a sample selected from the group consisting of biological samples, biological samples, environmental samples, and environmental samples.
[6]
The method according to any one of [1] to [4], wherein the specimen is derived from a sample selected from the group consisting of excrement samples, excrement-derived samples, vomit and vomit-derived samples.
[7]
The method according to any one of [1] to [6], wherein the surfactant is an anionic surfactant.
[8]
the anionic surfactant is alkyl sulfate, alkyl ether sulfate, docusate, sulfonate fluorosurfactant, alkylbenzene sulfonate, alkylaryl ether phosphate, alkyl ether phosphate, alkyl carboxylate, sodium lauroyl sarcosinate, carboxylate fluorosurfactant; The method of [7], wherein the anionic surfactant is one or more selected from the group consisting of sodium cholate and sodium deoxycholate.
[9]
The method of [7], wherein the anionic surfactant is an alkyl sulfate.
[10]
The method of [9], wherein the alkyl sulfate is sodium dodecyl sulfate or ammonium dodecyl sulfate.
[11]
The method according to any one of [1] to [10], wherein the surfactant has a concentration of 0.02 to 0.5% (w/v).
[12]
The method according to any one of [1] to [11], wherein the sample treatment liquid contains hydroxide.
[13]
The method of [12], wherein the hydroxide is sodium hydroxide or potassium hydroxide.
[14]
The method of [12] or [13], wherein the hydroxide concentration is 10 to 100 mM.
[15]
The method according to any one of [1] to [14], wherein the mixing ratio of the sample and the sample treatment liquid in step (1) is 1:3 to 6 as a volume ratio.
[16]
The method according to any one of [1] to [15], wherein the reverse transcriptase is selected from the group consisting of AMV reverse transcriptase, MMLV reverse transcriptase, HIV reverse transcriptase and variants thereof.
[17]
The method according to any one of [1] to [16], wherein the DNA polymerase is selected from the group consisting of Taq DNA polymerase, Tth DNA polymerase, KOD DNA polymerase, Pfu DNA polymerase and variants thereof.
[18]
The method according to any one of [1] to [17], wherein the step (4) is performed by real-time measurement.
[19]
The method according to any one of [1] to [18], wherein the steps (1) and (2) are performed at a temperature of 1 to 50°C.
[20]
In the step (4), the amplification curve of the RT-PCR product is measured using a fluorescence filter to determine whether the presence of the RNA virus in the sample is positive or negative, [1] to [19] ] The method according to any one of the above.

[21]
An RNA virus detection kit comprising a sample treatment solution containing one or more surfactants, and a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase.
[22]
The kit of [21], wherein the RNA virus is selected from the group consisting of norovirus, rotavirus, rhinovirus, coronavirus, human immunodeficiency virus, hepatitis C virus, Japanese encephalitis virus and dengue virus.
[23]
The kit of [21], wherein the RNA virus is norovirus.
[24]
The kit of [23], which determines whether the Norovirus genotype is genogroup I (GI) or genogroup II (GII).
[25]
The kit according to any one of [21] to [24], wherein the surfactant is an anionic surfactant.
[26]
the anionic surfactant is alkyl sulfate, alkyl ether sulfate, docusate, sulfonate fluorosurfactant, alkylbenzene sulfonate, alkylaryl ether phosphate, alkyl ether phosphate, alkyl carboxylate, sodium lauroyl sarcosinate, carboxylate fluorosurfactant; The kit of [25], which is at least one anionic surfactant selected from the group consisting of sodium cholate and sodium deoxycholate.
[27]
The kit of [25], wherein the anionic surfactant is an alkyl sulfate.
[28]
The kit of [27], wherein the alkyl sulfate is sodium dodecyl sulfate or ammonium dodecyl sulfate.
[29]
The kit according to any one of [21] to [28], further comprising an operation manual for the kit.

According to the present invention, RNA can be efficiently released from virus particles by directly mixing a sample such as stool containing RNA virus particles such as norovirus with a sample treatment solution containing one or more surfactants. can. For this reason, the steps that were conventionally required, that is, suspending the sample in water or a buffer solution, extracting the centrifugal supernatant from the resulting suspension by centrifugation, and adding the sample treatment solution to the extracted centrifugal supernatant This shortens the RNA release step of leaving the cells to stand and allows rapid detection of RNA viruses by RT-PCR. Furthermore, in the present invention, since the efficiency of RNA isolation from virus particles is high, virus detection sensitivity is high, and the virus excretion period can be detected with high accuracy. Therefore, it is useful for detecting subclinical infections, especially for identifying infected patients who are recovering from infection.

The present invention provides a method of detecting RNA viruses in a specimen. The method comprises
(1) mixing a specimen with a specimen treatment liquid containing one or more surfactants to form a suspension;
(2) extracting the centrifugal supernatant from the suspension produced in step (1) by centrifugation;
(3) mixing the centrifugal supernatant extracted in step (2) with a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase to perform RT-PCR;
(4) detecting the RT-PCR product;
include.

In the present invention, the RNA virus to be detected in the sample is a virus having RNA as a genome, and is a coronavirus having an envelope that is a membrane composed of a lipid bilayer, human immunodeficiency virus, hepatitis C virus, and Japanese encephalitis. Viruses such as dengue virus, non-enveloped norovirus, rotavirus, rhinovirus and the like, but are not limited thereto. Since the envelope is mainly composed of lipids, it is easily destroyed by organic solvents such as alcohol and surfactants. Resistant to

Specimens in the present invention include biological samples, biological-derived samples, environmental samples, environmental-derived samples, and the like. Biological samples include animal and plant tissues including midgut glands of shellfish and body fluids such as blood, saliva, nasal secretions, and tissue secretions. In particular, shellfish are regarded as the most important causative foods for food poisoning caused by norovirus. Biological samples include those obtained by subjecting the above biological samples to a treatment such as sonication. Environmental samples include any sample including air, soil, dust, water, and the like. Examples of environmental samples include those obtained by subjecting the environmental samples to processing such as sonication.

In another embodiment of the present invention, specimens include stool samples, stool-derived samples, vomit and vomit-derived samples. Excreta samples and vomit samples can be used as they are, or may be suspended in distilled water, physiological saline, or buffer solutions at, for example, 10% (w/v) to form emulsions. Examples of the buffer include, but are not limited to, phosphate buffer, Tris buffer, borate buffer, and Good buffer such as HEPES. The sample emulsion may be centrifuged at 10,000 to 12,000 rpm for 2 to 20 minutes to remove contaminants such as intestinal bacteria, and the centrifugal supernatant may be used as a sample. Feces-derived and vomit-derived samples include swabs. Wipe samples are used to confirm virus contamination. Fingers, tableware, cutting boards, kitchen knives, cooking equipment, toilet equipment, housing equipment, etc. were wiped with cotton swabs, cut cotton, etc., and eluted in phosphate buffer. It is. The obtained eluate is subjected to ultracentrifugation, and the centrifugal sediment suspended or dissolved can be used as a specimen (Yoshiko Munemura et al., Food Hygiene Journal, Vol. 58, No. 4, pp. 201-204, 2017). .

Step (1) of the present invention is a step of mixing a sample with a sample treatment liquid containing one or more surfactants. As used herein, "surfactant" is a general term for substances that act on the interface between substances to change their properties. A surfactant has a structure having both a hydrophilic portion and a hydrophobic portion in the molecule. Surfactants are classified into anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants. Anionic surfactants include alkyl sulfates, alkyl ether sulfates, docusate, sulfonate fluorosurfactants, alkylbenzene sulfonates, alkylaryl ether phosphates, alkyl ether phosphates, alkyl carboxylates, sodium lauroyl sarcosinate, carboxylate fluorosurfactants, Examples include, but are not limited to, sodium cholate and sodium deoxycholate. Preferred alkyl sulfates are Sodium Dodecyl Sulfate (SDS) and Ammonium Dodecyl Sulfate, more preferably Sodium Dodecyl Sulfate. Sodium dodecyl sulfate is also called Sodium Lauryl Sulfate (SLS). Cationic surfactants include, but are not limited to, ethyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, and the like. Amphoteric surfactants include, but are not limited to, betaines and alkylamino fatty acid salts. Nonionic surfactants include nonylphenoxypolyethoxyethanol (NP-40), polyoxyethylene sorbitan monooleate (Tween (registered trademark) 80), polyoxyethylene pt-octylphenol (Triton X-100 (registered Trademark)), etc., but are not limited to these.

When a surfactant is added to an aqueous solution at a certain concentration or higher, surfactant monomers aggregate to form micelles. The concentration at which surfactants form micelles is called the critical micelle concentration. In an aqueous solution, the hydrophobic regions of proteins and lipids are incorporated into the hydrophobic regions inside the surfactant micelles, so that the proteins and lipids are solubilized. In RNA virus particles, the protein shell, the capsid, and the lipid envelope are solubilized, denatured, or destroyed in the presence of a surfactant above the critical micelle concentration. As a result, the capsid-encapsulated RNA is more likely to be exposed in an aqueous solution. The critical micelle concentration of the surfactant varies depending on the type of surfactant, but in order to efficiently expose the viral RNA, the concentration of the surfactant in the sample treatment solution should be 0.02 to 0.5% (w /v), more preferably 0.05 to 0.3% (w/v), even more preferably 0.2% (w/v).

The mixing ratio of the sample and the sample-treated solution is preferably 1:3 to 6, more preferably 1:4 in terms of volume ratio. By mixing the specimen and the specimen treatment liquid containing the surfactant, the concentration of the surfactant in the mixed liquid decreases, but the above concentration of the surfactant maintains the critical micelle concentration.

In one embodiment of the present invention, the sample treatment liquid contains hydroxide. As used herein, the term “hydroxide” refers to a substance in which metal ions as cations and hydroxide ions (OH ⁻ ) as anions are ionic bonded. The metals are alkali metals or alkaline earth metals. Examples of hydroxides include lithium hydroxide, sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide and barium hydroxide, with sodium hydroxide and potassium hydroxide being preferred. Hydroxides are strongly basic and generate hydroxide ions when dissolved in water, so they are also called alkalis. Hydroxide changes the charge state of dissociable amino acids such as aspartic acid and glutamic acid in protein molecules in aqueous solution, thereby denaturing proteins. Due to this action, alkaline treatment of RNA virus particles results in disruption of the capsid. As a result, the capsid-encapsulated RNA is more likely to be exposed in an aqueous solution. In order to efficiently expose the viral RNA, the hydroxide concentration in the specimen treatment solution is preferably 10-100 mM, more preferably 40-60 mM, and even more preferably 50 mM.

In order to solubilize, denature, or destroy the capsid and lipid envelope and to efficiently expose the viral RNA, it is preferable that the sample treatment solution contains both a surfactant and a hydroxide.

The step (2) of the present invention is a step of mixing the sample with the sample-treated liquid and extracting the centrifugal supernatant from the resulting suspension by centrifugation. Such centrifugation can remove insoluble matter from the suspension. The centrifugation conditions are not particularly limited as long as insoluble matter can be removed, but are preferably 8000 to 12000 rpm for 2 to 10 minutes, preferably 10000 rpm for 5 minutes. In order to remove insoluble matters, other than centrifugation, for example, filtration using a filtration filter with a pore size of 0.22 to 0.45 μm may be performed.

In the steps (1) and (2) of the present invention for efficiently exposing viral RNA from the capsid and extracting it as a centrifugal supernatant, temperature control is not particularly required, but can be carried out at 1 to 50°C. Preferably, it is more preferably carried out at a room temperature of 1 to 30°C.

Step (3) of the present invention is a step of mixing the centrifugation supernatant obtained in step (2) with a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase and performing RT-PCR. Among the surfactants contained in the centrifugation supernatant obtained in step (2), SDS in particular has a strong denaturing effect on proteins. Therefore, if SDS is brought into the step (3) at a high concentration, it may inhibit the enzymatic activities of the reverse transcriptase and DNA polymerase contained in the one-step RT-PCR reaction solution, preventing the RT-PCR from proceeding. be. Similarly, if the concentration of hydroxide contained in the centrifugal supernatant obtained in step (2) is high and is brought into step (3), it will lead to a decrease in the enzyme activity due to high pH. Therefore, the mixing ratio of the centrifugation supernatant obtained in step (2) and the one-step RT-PCR reaction solution is preferably 1:2 to 6, more preferably 1:4 in terms of volume ratio.

In step (3) of the present invention, 1-step RT-PCR is employed in order to analyze multiple samples in a short time. In the one-step RT-PCR reaction solution, reverse transcriptase and DNA polymerase are mixed in advance, and reverse transcription reaction (single-strand cDNA synthesis) and PCR can be performed in the same vessel.

The reverse transcriptase contained in the one-step RT-PCR reaction solution is an enzyme that generates a single-stranded complementary DNA (cDNA) using viral RNA as a template, and is not particularly limited as long as it catalyzes the reverse transcription reaction. , Avian Myeloblastosis Virus (AMV), Moloney Murine Leukemia Virus (M-MLV) and Human Immunodeficiency Virus (HIV). DNA polymerases as well as variants thereof can be used. A DNA polymerase that also has reverse transcriptase activity can also be used. Such DNA polymerases are exemplified by Taq and Tth below.

The DNA polymerase contained in the one-step RT-PCR reaction solution is a thermostable DNA polymerase derived from thermophilic bacteria, and Taq, Tth, KOD, Pfu and variants thereof can be used, but are limited to these. not. A hot start DNA polymerase may be used to avoid non-specific amplification by the DNA polymerase. A hot-start DNA polymerase is, for example, a DNA polymerase to which an anti-DNA polymerase antibody is bound or a DNA polymerase in which the enzyme active site is heat-sensitive chemically modified. It is an enzyme that converts

The one-step RT-PCR reaction solution contains all the components for the reverse transcription reaction and PCR to be performed under suitable conditions. The components include at least the reverse transcriptase, the reverse transcription reaction primer, the thermostable DNA polymerase, the PCR primer, a dNTP mix (deoxyribonucleotide 5'-triphosphate; a mixture consisting of dATP, dGTP, dCTP and dTTP) and a buffer. . In one embodiment of the invention, the reaction liquid contains Tris and magnesium ions. An RNase inhibitor can also be added to the reaction solution. A primer specific to the target RNA sequence, an oligo(dT) primer, or a random primer can be used as the reverse transcription reaction primer. As PCR primers, a pair of primers (forward and reverse) specific to the sequence of cDNA generated by reverse transcription reaction is used. The PCR primers may be identical to the reverse transcription reaction primers specific for the target RNA sequence. In addition, two or more types of PCR primers may be added to the one-step RT-PCR reaction solution according to the number of DNA regions to be amplified, that is, the number of target sequences. As a composition containing the above components, reagents included in a norovirus detection reagent kit (probe method) (Shimadzu Corporation, product number 241-09325-91 or 241-09325-92) were mixed according to the kit instruction manual RT - PCR reactions can be used.

When detecting norovirus RNA, for example, by using the PCR primers described in Patent Documents 1 and 2, Non-Patent Document 3, and JP-A-2018-78806, Genogroup I (GI) and Genogroup I (GI) in norovirus genotypes Group II (GII) can be detected, but is not limited to these. Since the norovirus detection reagent kit (probe method) contains the PCR primers described in Non-Patent Document 3, these can be used.

A person skilled in the art can easily set the reaction temperature conditions for the reverse transcription reaction in RT-PCR and the PCR conditions (temperature, time and number of cycles).

Step (4) of the present invention is a step of detecting products from the RT-PCR performed in step (3). In one embodiment of the invention, PCR products are detected by real-time measurements. When the real-time measurement is performed, the RT-PCR in step (3) and the step of detecting the RT-PCR product in step (4) are performed in the same container.

Real-time measurement of PCR products is also called real-time PCR. In real-time PCR, PCR amplification products are usually detected by fluorescence. Fluorescence detection methods include a method using an intercalating fluorescent dye and a method using a fluorescently labeled probe. As an intercalating fluorescent dye, SYBR® Green I is used, but is not limited thereto. The intercalating fluorescent dye binds to double-stranded DNA synthesized by PCR and emits fluorescence upon irradiation with excitation light. By measuring this fluorescence intensity, the production amount of the PCR amplification product can be measured.

Fluorescently labeled probes include, but are not limited to, TaqMan probes, Molecular Beacons, cycling probes, and the like. TaqMan probes are oligonucleotides modified at the 5' end with a fluorescent dye and at the 3' end with a quencher substance. TaqMan probes specifically hybridize to template DNA in the annealing step of PCR, but the presence of a quencher on the probe suppresses the generation of fluorescence even when irradiated with excitation light. In the subsequent elongation reaction step, when the TaqMan probe hybridized to the template DNA is degraded by the 5'→3' exonuclease activity of Taq DNA polymerase, the fluorescent dye is released from the probe and fluorescence is generated by the quencher. is released and emits fluorescence. By measuring this fluorescence intensity, the production amount of the amplification product can be measured. Said fluorescent dyes include, but are not limited to, FAM, ROX, Cy5. Said quenchers include, but are not limited to TAMRA® and MGB. In order to discriminately detect two or more types of DNA target sequences, PCR is performed using two or more types of oligonucleotide probes (eg, TaqMan probes) each bound with a different fluorescent dye.

In step (4), the amplification curve of the RT-PCR product is measured using a fluorescent filter corresponding to the fluorescent dye used. If the fluorescence intensity increases according to the number of PCR cycles, the presence of the RNA virus to be analyzed in the sample is determined to be positive, while if the fluorescence intensity does not increase in PCR, it is determined to be negative. .

In one embodiment of the present invention, an RNA virus detection kit is provided that includes a sample treatment solution containing one or more surfactants, and a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase.

Claims (29)

A method for detecting an RNA virus in a specimen, comprising:
(1) mixing a specimen with a specimen treatment liquid containing one or more surfactants to form a suspension;
(2) extracting the centrifugal supernatant from the suspension produced in step (1) by centrifugation;
(3) mixing the centrifugal supernatant extracted in step (2) with a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase to perform RT-PCR;
(4) detecting the RT-PCR product;
method including. 2. The method of claim 1, wherein said RNA virus is selected from the group consisting of norovirus, rotavirus, rhinovirus, coronavirus, human immunodeficiency virus, hepatitis C virus, Japanese encephalitis virus and dengue virus. 2. The method of claim 1, wherein said RNA virus is Norovirus. 4. The method of claim 3, wherein the Norovirus genotype is Genogroup I (GI) or Genogroup II (GII). The method according to any one of claims 1 to 4, wherein the specimen is derived from a sample selected from the group consisting of biological samples, biological samples, environmental samples, and environmental samples. The method according to any one of claims 1 to 4, wherein the specimen is derived from a sample selected from the group consisting of a feces sample, a feces-derived sample, vomit, and a vomit-derived sample. The method of any one of claims 1-6, wherein the surfactant is an anionic surfactant. the anionic surfactant is alkyl sulfate, alkyl ether sulfate, docusate, sulfonate fluorosurfactant, alkylbenzene sulfonate, alkylaryl ether phosphate, alkyl ether phosphate, alkyl carboxylate, sodium lauroyl sarcosinate, carboxylate fluorosurfactant; 8. The method of claim 7, wherein the one or more anionic surfactants are selected from the group consisting of sodium cholate and sodium deoxycholate. 8. The method of claim 7, wherein said anionic surfactant is an alkyl sulfate. 10. The method of claim 9, wherein the alkyl sulfate is sodium dodecyl sulfate or ammonium dodecyl sulfate. The method of any one of claims 1-10, wherein the surfactant concentration is 0.02-0.5% (w/v). The method according to any one of claims 1 to 11, wherein the sample treatment liquid contains hydroxide. 13. The method of claim 12, wherein the hydroxide is sodium hydroxide or potassium hydroxide. 14. A method according to claim 12 or 13, wherein the hydroxide concentration is between 10 and 100 mM. 15. The method according to any one of claims 1 to 14, wherein the mixing ratio of the specimen and the specimen treatment liquid in the step (1) is 1:3 to 6 as a volume ratio. 16. The method of any one of claims 1-15, wherein the reverse transcriptase is selected from the group consisting of AMV reverse transcriptase, MMLV reverse transcriptase, HIV reverse transcriptase and variants thereof. 17. The method of any one of claims 1-16, wherein the DNA polymerase is selected from the group consisting of Taq DNA polymerase, Tth DNA polymerase, KOD DNA polymerase, Pfu DNA polymerase and variants thereof. A method according to any one of claims 1 to 17, wherein said step (4) is performed by real-time measurement. The method according to any one of claims 1 to 18, wherein said steps (1) and (2) are performed at a temperature of 1 to 50°C. The method of claims 1 to 19, wherein in the step (4), an amplification curve of the RT-PCR product is measured using a fluorescence filter to determine whether the presence of the RNA virus in the specimen is positive or negative. A method according to any one of paragraphs. An RNA virus detection kit comprising a sample treatment solution containing one or more surfactants, and a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase. 22. The kit of claim 21, wherein said RNA virus is selected from the group consisting of norovirus, rotavirus, rhinovirus, coronavirus, human immunodeficiency virus, hepatitis C virus, Japanese encephalitis virus and dengue virus. 22. The kit of claim 21, wherein said RNA virus is Norovirus. 24. The kit of claim 23, wherein the Norovirus genotype is determined to be Genogroup I (GI) or Genogroup II (GII). The kit according to any one of claims 21-24, wherein the surfactant is an anionic surfactant. the anionic surfactant is alkyl sulfate, alkyl ether sulfate, docusate, sulfonate fluorosurfactant, alkylbenzene sulfonate, alkylaryl ether phosphate, alkyl ether phosphate, alkyl carboxylate, sodium lauroyl sarcosinate, carboxylate fluorosurfactant; 26. The kit of claim 25, which is one or more anionic surfactants selected from the group consisting of sodium cholate and sodium deoxycholate. 26. The kit of claim 25, wherein said anionic surfactant is an alkyl sulfate. 28. The kit of claim 27, wherein said alkyl sulfate is sodium dodecyl sulfate or ammonium dodecyl sulfate. The kit according to any one of claims 21 to 28, further comprising kit operating instructions.