JP2020080806A - Methods for detecting rna viruses - Google Patents

Abstract

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

Description

TECHNICAL FIELD The present invention relates to a method for detecting an RNA virus 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 virus by mixing a sample with a sample treatment solution containing a surfactant and further adding an RT-PCR reaction solution, and a kit for carrying out the method.

The RNA virus is a virus having RNA as a genome, and includes coronavirus having an envelope that is a membrane composed of a lipid bilayer, human immunodeficiency virus, hepatitis C virus, Japanese encephalitis virus, dengue virus, and norovirus having no envelope. , Rotavirus, rhinovirus, etc., many of which are pathogenic.

Norovirus is a virus belonging to the human calicivirus family and has a single-stranded RNA of about 7,000 bases in its genome. This virus is also called Small Round Structured Virus (SRSV) according to the morphological classification observed under an electron microscope, and has also been called the generic name of Norwalk-like virus. Is. Viruses belonging to Norovirus are classified into two gene groups, Genogroup (GI) and Genogroup II (GII), and further classified into 14 and 17 or more genotypes, respectively.

Infection of humans with Norovirus causes acute gastrointestinal inflammation such as vomiting and diarrhea. Approximately half of all food poisoning patients in Japan are caused by norovirus, about 70% of which occur in November to February, and norovirus is known as a causative virus of winter type gastroenteritis and food poisoning. Food poisoning due to norovirus is mainly caused by contamination of food through the cook. Norovirus is highly infectious and is prone to outbreaks such as large-scale food poisoning. The route of infection for humans is mainly oral infection. Representative sources of infection include feces or vomitus of infected persons and articles directly or indirectly contaminated therewith, and foods such as oysters or other bivalves contaminated with norovirus. Therefore, it is important to identify patients infected with Norovirus and contaminants from the virus in order to prevent the spread of viral infection.

As a virus test for detecting infection or contamination by a virus, immunological assay methods for detecting viral antigens and viral gene amplification methods are used (Patent Documents 1 to 3, Non-Patent Document 1). As a method for highly sensitively measuring Norovirus, there is a method of amplifying Norovirus RNA by RT-PCR and measuring the amount of the amplified product. For example, detection of norovirus by the RT-PCR method and quantitative detection of norovirus by the real-time PCR method are widely performed in accordance with the notification (Non-patent documents 2 and 3) by the Food Safety Division, Food Safety Department, Ministry of Health, Labor and Welfare. ing.

RNA virus particles have a basic structure in which a core composed of an RNA genome and a protein is enclosed in a protein shell called a capsid. Therefore, in order to detect viral RNA by the gene amplification method, it is necessary to extract RNA from viral particles. To detect norovirus in feces as a sample, for example, a fecal sample is suspended in distilled water or physiological saline at a concentration of 5 to 10% (w/v), and commercially available viral RNA is extracted from the centrifugation supernatant. RNA is extracted and purified using a kit (for example, QIAamp (registered trademark) Viral RNA Mini, QIAGEN) (Non-patent document 2). However, the detection process of performing RT-PCR after multi-step RNA extraction/purification operations is complicated. For this reason, a simple detection method in which the fecal suspension is mixed with the sample treatment solution and the shell protein is removed by heat treatment for a short time to release the internal RNA 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 fecal suspension and the sample treatment liquid, the reaction container is closed with a lid to prevent bumping or evaporation of the mixture, and after the heat treatment, the lid is removed and the RT-PCR reaction liquid is added. It takes time to do. In order to improve this point, there has been proposed a method of detecting a virus by RT-PCR by mixing a sample with a chaotropic agent such as a guanidine salt, without performing heat treatment (Patent Document 4).

WO2002/029119 WO2002/029120 Japanese Patent Laid-Open No. 2004-301684 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, Food Safety Department, Food Safety Department, Monitoring and Safety Division, Food Safety Audit No. 1105001 (November 5, 2003) Attachment “About detection method of norovirus”, final revision: Food Safety Audit 0514004 (May 2007) 14th) Ministry of Health, Labor and Welfare, Food Safety Department, Food Safety Department, Surveillance and Safety Division, Food Safety Audit No. 1105001 (November 5, 2003) Attachment “About detection method of norovirus”, final revision: Food Safety Audit 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 virus. Specifically, RNA extraction from RNA virus particles such as norovirus is performed without heat treatment using one or more kinds of surfactants, and the subsequent virus detection operation by RT-PCR reaction of released RNA is simplified. To provide a way to do it.

The object of the present invention is achieved by the following inventions.
[1]
A method for detecting RNA virus in a sample, comprising:
(1) A step of mixing a sample with a sample processing liquid containing one or more surfactants to form a suspension,
(2) A step of extracting a centrifugal supernatant by centrifugation from the suspension generated in the step (1),
(3) A step of mixing the centrifugal supernatant extracted in step (2) with a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase, and performing RT-PCR, and
(4) detecting the RT-PCR product,
Including the method.
[2]
The method according to [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 according to [1], wherein the RNA virus is Norovirus.
[4]
The method according to [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 sample 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 sample is derived from a sample selected from the group consisting of excrement samples, excrement-derived samples, vomitus and vomitus-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 an alkyl sulfate, an alkyl ether sulfate, docusate, a sulfonate fluoro surfactant, an alkyl benzene sulfonate, an alkyl aryl ether phosphate, an alkyl ether phosphate, an alkyl carboxylate, sodium lauroyl sarcosine, a carboxylate fluoro surfactant, The method according to [7], which is one or more anionic surfactants selected from the group consisting of sodium cholate and sodium deoxycholate.
[9]
The method according to [7], wherein the anionic surfactant is an alkyl sulfate.
[10]
The method according to [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 concentration of the surfactant is 0.02 to 0.5% (w/v).
[12]
The method according to any one of [1] to [11], wherein the sample treatment liquid contains a hydroxide.
[13]
The method according to [12], wherein the hydroxide is sodium hydroxide or potassium hydroxide.
[14]
The method according to [12] or [13], wherein the concentration of the hydroxide 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 the 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), an amplification curve of the RT-PCR product is measured using a fluorescent filter to determine whether the presence of RNA virus in the sample is positive or negative, [1] to [19 ] The method of any one of.
[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 according to [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 according to [21], wherein the RNA virus is Norovirus.
[24]
The kit according to [23], wherein the norovirus genotype is determined to be 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 an alkyl sulfate, an alkyl ether sulfate, docusate, a sulfonate fluoro surfactant, an alkyl benzene sulfonate, an alkyl aryl ether phosphate, an alkyl ether phosphate, an alkyl carboxylate, sodium lauroyl sarcosine, a carboxylate fluoro surfactant, The kit according to [25], which is one or more anionic surfactants selected from the group consisting of sodium cholate and sodium deoxycholate.
[27]
The kit according to [25], wherein the anionic surfactant is an alkyl sulfate.
[28]
The kit according to [27], wherein the alkyl sulfate is sodium dodecyl sulfate or ammonium dodecyl sulfate.
[29]
Further, the kit according to any one of [21] to [28], which further includes a procedure manual for the kit.

According to the present invention, RNA can be efficiently released from virus particles by directly mixing a sample such as feces containing RNA virus particles such as Norovirus with a sample treatment solution containing one or more surfactants. it can. For this reason, a conventionally required step, namely, suspending the sample in water or a buffer solution, extracting the centrifugation supernatant from the resulting suspension by centrifugation, and adding the sample treatment liquid to the extracted centrifugation supernatant Then, the RNA releasing step of leaving it for standing is shortened, and the RNA virus can be promptly detected by RT-PCR. Furthermore, in the present invention, since the efficiency of RNA release from virus particles is high, the virus detection sensitivity is high and the virus excretion period can be accurately detected. Therefore, it is useful for detecting subclinical infection, particularly for identifying infected patients in a convalescent stage after infection.

The present invention provides a method for detecting RNA virus in a sample. The method comprises the steps of (1) mixing a sample with a sample treatment liquid containing one or more surfactants to form a suspension;
(2) a step of extracting a centrifugal supernatant by centrifugation from the suspension generated in the step (1),
(3) A step of mixing the centrifugal supernatant extracted in step (2) with a 1-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase, and performing RT-PCR, and
(4) detecting the RT-PCR product,
Including.

In the present invention, the RNA virus to be detected in the sample is a virus having RNA as a genome and has a coronavirus having an envelope that is a membrane composed of a lipid bilayer, human immunodeficiency virus, hepatitis C virus, Japanese encephalitis. Examples include, but are not limited to, viruses, dengue viruses, and non-enveloped noroviruses, rotaviruses, rhinoviruses, and the like. Since the main component of the envelope is lipid, it is easily destroyed by an organic solvent such as alcohol or a surfactant, but RNA viruses such as Norovirus that do not have such an envelope are generally treated with an organic solvent or a surfactant. Shows resistance to.

Examples of the sample in the present invention include biological samples, biological samples, environmental samples and environmental samples. Biological samples include animal and plant tissues including the midgut glands of shellfish and body fluids such as blood, saliva, nasal discharge, and tissue secretions. In particular, shellfish is regarded as the most important food as a causative food for food poisoning due to norovirus. The biological sample includes a sample obtained by subjecting the biological sample to treatment such as sonication. Examples of environmental samples include all samples including air, soil, dust, water and the like. The environment-derived sample includes a sample obtained by subjecting the environment sample to a treatment such as sonication.

In another embodiment of the present invention, the sample includes an excrement sample, an excrement-derived sample, a vomitus, a vomiting-derived sample, and the like. The excrement sample and vomiting sample can be used as they are, but may be suspended in distilled water, physiological saline or a buffer solution at, for example, 10% (w/v) to form an emulsion. The buffer solution is not particularly limited, and examples thereof include a phosphate buffer solution, a Tris buffer solution, a borate buffer solution, and a Good buffer solution such as HEPES. In order to remove contaminants such as intestinal bacteria, the emulsion of the sample may be centrifuged at 10000 to 12000 rpm for 2 to 20 minutes, and the centrifuged supernatant may be used as a sample. Excretion-derived and vomit-derived samples include wipe samples. The wiping sample was obtained by wiping fingers, tableware, cutting boards, knives, cooking equipment, toilet equipment, housing equipment, etc. with a cotton swab, cut cotton, etc. to elute in a phosphate buffer solution, etc. for the purpose of confirming virus contamination. It is a thing. The obtained eluate is subjected to ultracentrifugation and the suspension or solution of the centrifugal sediment can be used as a sample (Keiko Munemura et al., Journal of Food Hygiene, Vol. 58, No. 4, p. 201-204, 2017). ..

The step (1) of the present invention is a step of mixing the sample with a sample treatment liquid containing one or more kinds of surfactants. As used herein, the term “surfactant” is a general term for substances that act on the boundary surface of substances and change their properties. The 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. As the anionic surfactant, alkyl sulfate, alkyl ether sulfate, docusate, sulfonate fluoro surfactant, alkyl benzene sulfonate, alkyl aryl ether phosphate, alkyl ether phosphate, alkyl carboxylate, lauroyl sarcosine sodium, carboxylate fluoro surfactant, Examples include, but are not limited to, sodium cholate and sodium deoxycholate. As the alkyl sulfate, sodium dodecyl sulfate (SDS) and ammonium dodecyl sulfate are preferable, and sodium dodecyl sulfate is more preferable. Sodium dodecyl sulfate is also referred to as 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, betaine and alkylamino fatty acid salts. As the nonionic surfactant, nonylphenoxy polyethoxy ethanol (NP-40), polyoxyethylene sorbitan monooleate (Tween (registered trademark) 80), polyoxyethylene p-t-octylphenol (Triton X-100 (registered). Trademarks)) and the like, but are not limited thereto.

When a surfactant is added at a certain concentration or higher in an aqueous solution, the surfactant monomers aggregate to form micelles. The concentration at which the surfactant becomes micelle-forming is called the critical micelle concentration. In an aqueous solution, the hydrophobic region of the protein or lipid is incorporated into the hydrophobic region inside the surfactant micelle, and the protein or lipid is solubilized. In RNA virus particles, the envelope consisting of capsids and lipids that are protein shells is solubilized, denatured, or destroyed in the presence of a surfactant at a critical micelle concentration or higher. As a result, the RNA encapsulated in the capsid is likely to be exposed in the aqueous solution. Although the critical micelle concentration of the surfactant varies depending on the type of the surfactant, in order to efficiently expose the viral RNA, the concentration of the surfactant in the sample treatment liquid is 0.02 to 0.5% (w. /V) is preferable, 0.05 to 0.3% (w/v) is more preferable, and 0.2% (w/v) is further preferable.

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

In one embodiment of the present invention, the sample treatment liquid contains hydroxide. In the present specification, “hydroxide” refers to a substance in which a metal ion as a cation and a hydroxide ion (OH ⁻ ) as an anion are ionically bonded. The metal is an alkali metal or an alkaline earth metal. 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 when they are dissolved in water, they generate hydroxide ions, so they are also called alkalis. Hydroxide changes the charge state of dissociative amino acids such as aspartic acid and glutamic acid in a protein molecule in an aqueous solution to denature the protein. This action causes the capsid to be destroyed when the RNA virus particles are treated with alkali. As a result, the RNA encapsulated in the capsid is likely to be exposed in the aqueous solution. In order to efficiently expose viral RNA, the hydroxide concentration in the sample treatment solution is preferably 10 to 100 mM, more preferably 40 to 60 mM, and further preferably 50 mM.

In order to solubilize, denature, or destroy the envelope composed of capsids and lipids to efficiently expose viral RNA, it is preferable that a surfactant and a hydroxide coexist in the sample treatment liquid.

The step (2) of the present invention is a step of mixing the sample with the sample treatment liquid, and centrifuging the resulting suspension to extract the centrifugal supernatant. By such centrifugation, insoluble matter can be removed from the suspension. The centrifugation conditions are not particularly limited as long as the insoluble matter can be removed, but 8000 to 12000 rpm is 2 to 10 minutes, and 10000 rpm is preferably 5 minutes. In order to remove the insoluble matter, for example, filtration using a filtration filter having a pore size of 0.22 to 0.45 μm may be performed in addition to centrifugation.

In steps (1) and (2) of the present invention for efficiently exposing viral RNA from the capsid and extracting it as a centrifugal supernatant, it is not necessary to control the temperature, but it may be performed at 1 to 50°C. It is more preferable to carry out at room temperature of 1 to 30°C.

The step (3) of the present invention is a step of mixing the centrifugal supernatant obtained in the step (2) and a 1-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase and performing RT-PCR. Among the surfactants contained in the centrifugation supernatant obtained in step (2), SDS has a strong denaturing effect on proteins. Therefore, if SDS is brought to the step (3) at a high concentration, the enzymatic activities of the reverse transcriptase and the DNA polymerase contained in the 1-step RT-PCR reaction solution may be inhibited, and RT-PCR may not proceed. is there. Similarly, the hydroxide contained in the centrifugation supernatant obtained in step (2) also causes a decrease in the enzyme activity due to high pH when the concentration brought to step (3) is high. Therefore, the mixing ratio of the centrifugal supernatant obtained in the step (2) and the 1-step RT-PCR reaction solution is preferably 1:2 to 6 as a volume ratio, and more preferably 1:4.

In the step (3) of the present invention, one-step RT-PCR is adopted in order to analyze many samples in a short time. Reverse transcriptase and DNA polymerase are premixed in the 1-step RT-PCR reaction solution, and the reverse transcription reaction (single-stranded cDNA synthesis) and PCR can be performed in the same container.

The reverse transcriptase contained in the 1-step RT-PCR reaction solution is an enzyme that produces 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. Dependence of RNA Virus Derived from Avian Myeloblastosis Virus (AMV), Moloney Murine Leukemia Virus (M-MLV) and Human Immunodeficiency Virus (HIV) DNA polymerase as well as variants thereof can be used. Alternatively, a DNA polymerase having reverse transcriptase activity can also be used. Examples of such a DNA polymerase include Taq and Tth described below.

The DNA polymerase contained in the 1-step RT-PCR reaction solution is a thermostable bacterium-derived thermostable DNA polymerase, and Taq, Tth, KOD, Pfu and mutants thereof can be used, but are not limited thereto. Not done. A hot start DNA polymerase may be used to avoid non-specific amplification by the DNA polymerase. The 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 an enzyme active site is chemically modified by heat-sensitive chemical reaction. In PCR, the DNA polymerase is activated after the first denaturation step (90° C. or higher). It is an enzyme that is converted into.

The 1-step RT-PCR reaction solution contains all components for performing reverse transcription reaction and PCR under appropriate conditions. The components include at least the reverse transcriptase, reverse transcription reaction primer, thermostable DNA polymerase, PCR primer, dNTP mix (deoxyribonucleotide 5'-triphosphate; mixture of dATP, dGTP, dCTP and dTTP) and a buffer solution. .. In one embodiment of the present invention, the reaction liquid contains tris and magnesium ions. An RNA degrading enzyme inhibitor may be added to the reaction solution. As the reverse transcription reaction primer, a primer specific to the sequence of the target RNA, an oligo(dT) primer or a random primer can be used. As the PCR primers, a primer pair (forward and reverse) specific to the sequence of cDNA generated by the reverse transcription reaction is used. The PCR primer may be identical to the reverse transcription reaction primer specific for the sequence of the target RNA. Further, two or more kinds of PCR primers may be added to the one-step RT-PCR reaction solution depending on the number of DNA regions to be amplified, that is, target sequences. As a composition containing the above components, the reagents contained in a Norovirus detection reagent kit (probe method) (Shimadzu, product number 241-09325-91 or 241-09325-92) were mixed according to the kit instruction manual, and RT was added. -A PCR reaction solution 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 II in the norovirus genotype are used. (GII) can be detected, but is not limited thereto. Since the Norovirus detection reagent kit (probe method) includes the PCR primers described in Non-Patent Document 3, these can be used.

Those 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 the product from RT-PCR performed in step (3). In one embodiment of the invention, the PCR product is detected by real time measurement. When performing the real-time measurement, the RT-PCR of step (3) and the step of detecting the RT-PCR product of 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. The fluorescence detection method includes a method using an intercalating fluorescent dye and a method using a fluorescence-labeled probe. As the intercalating fluorescent dye, SYBR (registered trademark) Green I is used, but it is not limited thereto. The intercalating fluorescent dye binds to the double-stranded DNA synthesized by PCR and emits fluorescence upon irradiation with excitation light. By measuring this fluorescence intensity, the amount of PCR amplification product produced can be measured.

Fluorescently labeled probes include, but are not limited to, TaqMan probes, Molecular Beacon, cycling probes and the like. The TaqMan probe is an oligonucleotide modified at the 5'end with a fluorescent dye and at the 3'end with a quencher substance. The TaqMan probe specifically hybridizes to the 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 extension reaction step, when the TaqMan probe hybridized to the template DNA is decomposed by the 5′→3′ exonuclease activity of Taq DNA polymerase, the fluorescent dye is released from the probe and the fluorescence is generated by the quencher. Is suppressed and emits fluorescence. By measuring this fluorescence intensity, the amount of amplification product produced can be measured. Examples of the fluorescent dye include, but are not limited to, FAM, ROX, and Cy5. The quenchers include, but are not limited to, TAMRA® and MGB. In order to detect two or more types of DNA target sequences separately, PCR is performed using two or more types of oligonucleotide probes (for example, TaqMan probes) to which different fluorescent dyes are bound.

In step (4), an amplification curve of the RT-PCR product is measured using a fluorescent filter corresponding to the fluorescent dye used. When 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 when 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 which comprises a sample treatment solution containing one or more surfactants and a one-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase.

Claims (29)

A method for detecting RNA virus in a sample, comprising:
(1) A step of mixing a sample with a sample processing liquid containing one or more surfactants to form a suspension,
(2) a step of extracting a centrifugal supernatant by centrifugation from the suspension generated in the step (1),
(3) A step of mixing the centrifugal supernatant extracted in step (2) with a 1-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase, and performing RT-PCR, and
(4) detecting the RT-PCR product,
Including the method. The method according to claim 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. The method of claim 1, wherein the RNA virus is a Norovirus. 4. The method of claim 3, wherein the Norovirus genotype is Genogroup I (GI) or Genogroup II (GII). The method according to claim 1, 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 excrement samples, excrement-derived samples, vomitus and vomitus-derived samples. 7. The method according to any one of claims 1-6, wherein the surfactant is an anionic surfactant. The anionic surfactant is an alkyl sulfate, an alkyl ether sulfate, docusate, a sulfonate fluoro surfactant, an alkyl benzene sulfonate, an alkyl aryl ether phosphate, an alkyl ether phosphate, an alkyl carboxylate, sodium lauroyl sarcosine, a carboxylate fluoro surfactant, The method according to claim 7, which is one or more anionic surfactants selected from the group consisting of sodium cholate and sodium deoxycholate. The method of claim 7, wherein the 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 according to any one of claims 1 to 10, wherein the concentration of the surfactant is 0.02 to 0.5% (w/v). The method according to claim 1, wherein the sample treatment liquid contains a hydroxide. 13. The method of claim 12, wherein the hydroxide is sodium hydroxide or potassium hydroxide. The method according to claim 12 or 13, wherein the concentration of the hydroxide is 10 to 100 mM. The method according to any one of claims 1 to 14, wherein the mixing ratio of the sample and the sample 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 according to any one of claims 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. The method according to claim 1, wherein the step (4) is performed by real-time measurement. The method according to any one of claims 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 fluorescent filter to determine whether the presence of RNA virus in the sample is positive or negative. The method according to any one of items. 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 according to claim 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. 22. The kit of claim 21, wherein the RNA virus is Norovirus. The kit according to claim 23, which determines that the Norovirus genotype is Genogroup I (GI) or Genogroup II (GII). The kit according to any one of claims 21 to 24, wherein the surfactant is an anionic surfactant. The anionic surfactant is an alkyl sulfate, an alkyl ether sulfate, docusate, a sulfonate fluoro surfactant, an alkyl benzene sulfonate, an alkyl aryl ether phosphate, an alkyl ether phosphate, an alkyl carboxylate, sodium lauroyl sarcosine, a carboxylate fluoro surfactant, The kit according to 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 the anionic surfactant is an alkyl sulfate. 28. The kit of claim 27, wherein the alkyl sulfate is sodium dodecyl sulfate or ammonium dodecyl sulfate. 29. The kit according to any one of claims 21 to 28, further comprising a procedure manual for the kit.