RNA virus detection methods

Abstract

To provide a method for performing RNA extraction from RNA virus particles of a norovirus or the like by using one or more kinds of surfactant without heating treatment, and simply performing a subsequent virus detection operation of a free RNA by RT-PCR reaction, and a simpler detection method of an RNA virus by performing RNA extraction from RNA virus particles and RT-PCR in the same container without opening / closing a lid of the container.SOLUTION: The present invention relates to a method for detecting an RNA virus by reverse transcription-polymerase chain reaction (RT-PCR), and a kit for executing the method. More specifically, the invention relates to a method for detecting an RNA virus by mixing a sample with surfactant in the presence of hydroxide and furthermore by adding RT-PCR reaction liquid, and a kit for executing the method.SELECTED DRAWING: None

Description

[Technical Field]

[0001] 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 surfactant in the presence of hydroxide and then adding an RT-PCR reaction solution, and a kit for carrying out the method. [Background technology]

[0002] RNA viruses are viruses that have RNA as their genome and are classified into those with an envelope made of a lipid bilayer membrane, such as coronavirus, human immunodeficiency virus, hepatitis C virus, Japanese encephalitis virus, and dengue virus, as well as those without an envelope, such as norovirus, rotavirus, and rhinovirus, and many of them are pathogenic.

[0003] Noroviruses belong to the human Caliciviridae family and have a genome consisting of a single-stranded RNA of approximately 7,000 bases. Based on morphological classification observed under an electron microscope, they are also known as small round structured viruses (SRSVs) and have been given the genus name Norwalk-like viruses. Noroviruses are classified into two genogroups, genogroup GI (GI) and genogroup II (GII), which are further divided into 14 and 17 or more genotypes, respectively.

[0004] Norovirus infection in humans causes acute gastroenteritis symptoms such as vomiting and diarrhea. Approximately half of the annual food poisoning cases in Japan are caused by norovirus, with approximately 70% occurring between November and February. Norovirus is known to be the cause of winter-type gastroenteritis and food poisoning. Norovirus food poisoning primarily occurs through food contamination by food cooks. Norovirus is highly contagious and prone to mass outbreaks, including large-scale food poisoning. Human infection is primarily via oral transmission. Typical sources of infection include infected individuals' feces or vomit, items directly or indirectly contaminated by these, and foods such as oysters or other bivalve mollusks contaminated with norovirus. Therefore, identifying norovirus-infected patients and items contaminated by the virus is important for preventing the spread of viral infection.

[0005] Immunological assays for detecting viral antigens and viral gene amplification methods are used in virus tests to detect viral infections and contamination (Patent Documents 1 to 3, Non-Patent Document 1). A highly sensitive method for measuring norovirus is to amplify norovirus RNA by RT-PCR and measure the amount of the amplified product. For example, in accordance with notifications from the Surveillance and Safety Division, Food Safety Department, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor and Welfare (Non-Patent Documents 2 and 3), norovirus detection by RT-PCR and quantitative detection by real-time PCR are widely used.

[0006] RNA virus particles have a basic structure in which a core consisting of an RNA genome and protein is enclosed in a protein shell called a capsid. Therefore, in order to detect viral RNA using gene amplification methods, RNA must be extracted from the virus 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 5-10% (w / v), and RNA is extracted and purified from the centrifuged supernatant using a commercially available viral RNA extraction kit (e.g., QIAamp® Viral RNA Mini, QIAGEN) (Non-Patent Document 2). However, the detection process, which involves multiple steps of RNA extraction and purification followed by RT-PCR, is cumbersome. For this reason, a simple detection method has been proposed in which the fecal suspension is mixed with a sample treatment solution and briefly heat-treated to remove the shell protein, liberating the internal RNA, and then directly subjecting the liberated RNA to RT-PCR (Non-Patent Document 4). On the other hand, heat treatment of a mixture of a fecal suspension and a specimen treatment solution requires the laborious task of sealing the reaction vessel with a lid to prevent the mixture from boiling or evaporating, and then removing the lid after heat treatment to add the RT-PCR reaction solution. To address this issue, a method has been proposed for detecting viruses by RT-PCR without heat treatment by mixing the specimen with a chaotropic agent such as a guanidine salt (Patent Document 4). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. WO2002 / 029119 [Patent Document 2] International Publication No. WO2002 / 029120 [Patent Document 3] Japanese Patent Application Laid-Open No. 2004-301684 [Patent Document 4] Japanese Patent Application Publication No. 2017-209036 [Non-patent literature]

[0008] [Non-Patent Document 1] Kageyama T, et al. Broadly reactive and highly sensitive assay sensitive for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. J Clin Microbiol. 2003 Apr;41(4):1548-57. [Non-patent document 2] Ministry of Health, Labour and Welfare, Food Safety Inspection Division, Food Safety Department, Pharmaceuticals and Food Safety Bureau, Food Safety Inspection Notification No. 1105001 (November 5, 2003) with an attachment "Detection Methods for Norovirus"; last revised: Food Safety Inspection Notification No. 0514004 (May 14, 2007) [Non-patent document 3] Ministry of Health, Labour and Welfare, Food Safety Inspection Division, Food Safety Department, Pharmaceuticals and Food Safety Bureau, Food Safety Inspection Notification No. 1105001 (November 5, 2003) with an attachment "Detection Methods for Norovirus"; last revised: Food Safety Inspection Notification No. 1022-1 (October 22, 2013) [Non-patent document 4] 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. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a simple method for detecting RNA viruses. Specifically, the present invention provides a method for easily extracting RNA from RNA virus particles such as norovirus using one or more surfactants without heat treatment, followed by virus detection by RT-PCR of the released RNA. Furthermore, the present invention provides a simpler method for detecting RNA viruses by performing RNA extraction from RNA virus particles and RT-PCR in the same container without opening or closing the container lid. [Means for solving the problem]

[0010] The object of the present invention is achieved by the following inventions. [1] 1. A method for detecting an RNA virus in a sample, comprising: (1) A step of suspending a specimen in distilled water, physiological saline, or a buffer solution; (2) extracting the supernatant from the suspension obtained in step (1); (3) mixing the centrifugal supernatant extracted in step (2) with a sample treatment solution containing one or more surfactants; (4) mixing the mixture obtained in step (3) with a one-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase, and performing RT-PCR; and (5) detecting the RT-PCR product; A method comprising:

[0011] [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.

[0012] [3] The method according to [1], wherein the RNA virus is norovirus. [4] The method described in [3], wherein the norovirus genotype is genogroup I (GI) or genogroup II (GII).

[0013] [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 a biological sample, a biologically derived sample, an environmental sample, and an environmentally derived sample. [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 a fecal sample, a fecal-derived sample, vomit, and a vomit-derived sample. [7] The method according to any one of [1] to [6], wherein the surfactant is an anionic surfactant.

[0014] [8] 8. The method according to claim 7, wherein the anionic surfactant is one or more anionic surfactants selected from the group consisting of alkyl sulfates, alkyl ether sulfates, docusate, sulfonate fluorosurfactants, alkyl benzene sulfonates, alkyl aryl ether phosphates, alkyl ether phosphates, alkyl carboxylates, sodium lauroyl sarcosinate, carboxylate fluorosurfactants, 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.

[0015]

[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 solution 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.

[0016]

[15] The method according to any one of [1] to

[14] , wherein the mixing ratio of the centrifugal supernatant to the specimen treatment solution in the step (3) is 1:3 to 1:6 in terms of 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 mutants 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 mutants thereof.

[18] The method according to any one of [1] to

[17] , wherein the step (5) is carried out by real-time measurement.

[19] The method according to any one of [1] to

[18] , wherein the step (3) is carried out at a temperature of 1 to 60°C.

[20] The method according to any one of [1] to

[19] , wherein steps (3) to (5) are carried out in the same container. 〔twenty one〕 The method according to any one of [1] to

[20] , wherein in step (5), an amplification curve of the RT-PCR product is measured using a fluorescent filter, and the presence of an RNA virus in the sample is determined to be positive or negative.

[0017] 〔twenty two〕 A kit for detecting RNA viruses, comprising a sample treatment solution containing one or more surfactants, and a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase. 〔twenty three〕 The kit according to

[22] , 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. 〔twenty four〕 The kit according to

[22] , wherein the RNA virus is norovirus. 〔twenty five〕 The kit according to

[24] , which determines whether the norovirus genotype is genogroup I (GI) or genogroup II (GII).

[0018]

[26] The kit according to any one of

[22] to

[25] , wherein the surfactant is an anionic surfactant.

[27] The kit according to

[26] , wherein the anionic surfactant is one or more anionic surfactants selected from the group consisting of alkyl sulfates, alkyl ether sulfates, docusate, sulfonate fluorosurfactants, alkyl benzene sulfonates, alkyl aryl ether phosphates, alkyl ether phosphates, alkyl carboxylates, sodium lauroyl sarcosinate, carboxylate fluorosurfactants, sodium cholate, and sodium deoxycholate.

[28] The kit according to

[26] , wherein the anionic surfactant is an alkyl sulfate.

[29] The kit according to

[28] , wherein the alkyl sulfate is sodium dodecyl sulfate or ammonium dodecyl sulfate.

[30] The kit according to any one of

[22] to

[29] , further comprising an operating manual for the kit. [Effects of the Invention]

[0019] According to the present invention, RNA can be efficiently liberated from virus particles without heat treatment by mixing the centrifugal supernatant of a sample suspension containing RNA virus particles such as norovirus with a sample treatment solution containing one or more surfactants. Therefore, the RNA liberation process and the subsequent addition of an RT-PCR reaction solution for detecting the presence of the virus can be performed consecutively in the same container, allowing for convenient detection of RNA viruses. Furthermore, the present invention provides high efficiency in liberating RNA from virus particles, resulting in high virus detection sensitivity and accurate detection of the period of viral shedding. Therefore, the present invention is useful for detecting asymptomatic infections, particularly for identifying infected patients in the recovery phase after infection. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides a method for detecting an RNA virus in a specimen, comprising the steps of (1) suspending the specimen in distilled water, physiological saline, or a buffer solution, (2) extracting the supernatant from the suspension obtained in step (1), (3) mixing the supernatant extracted in step (2) with a specimen treatment solution containing one or more surfactants, (4) mixing the mixture obtained in step (3) with a one-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase, and performing RT-PCR, and (5) detecting the RT-PCR product.

[0021] In the present invention, RNA viruses to be detected in samples are viruses that have RNA as their genome, and include, but are not limited to, coronaviruses, human immunodeficiency viruses, hepatitis C viruses, Japanese encephalitis viruses, dengue viruses, etc., which have an envelope made of a lipid bilayer membrane, as well as noroviruses, rotaviruses, rhinoviruses, etc. Since the main component of the envelope is lipid, it is easily destroyed by organic solvents such as alcohols and surfactants, but such non-enveloped RNA viruses such as noroviruses are generally resistant to organic solvents and surfactants.

[0022] Specimens in the present invention include biological samples, biologically derived samples, environmental samples, and environmentally derived samples. Biological samples include animal and plant tissues, including the midgut gland of shellfish, and body fluids, such as blood, saliva, nasal secretions, and tissue secretions. Shellfish, in particular, are considered the most important food source for norovirus food poisoning. Biologically derived samples include those obtained by treating the above biological samples with, for example, sonication. Environmental samples include all samples, including air, soil, dust, and water. Environmentally derived samples include those obtained by treating the above environmental samples with, for example, sonication.

[0023] In another embodiment of the present invention, the specimen may include a fecal sample, a fecal-derived sample, vomit, and a vomit-derived sample. Fecal and vomit samples may be used as they are, or in step (1), they may be suspended in distilled water, physiological saline, or a buffer solution, for example, at a concentration of 10% (w / v), to form an emulsion. Examples of the buffer solution include, but are not limited to, phosphate buffer, Tris buffer, borate buffer, and Good's buffer such as HEPES. To remove contaminants such as enterobacteria, the emulsion may be centrifuged at 10,000 to 12,000 rpm for 2 to 20 minutes in step (2), and the supernatant may be used as the specimen. Fecal and vomit-derived samples include swab samples. Swab samples are prepared by wiping hands, tableware, cutting boards, knives, kitchen equipment, toilet equipment, and household fixtures with cotton swabs or cotton wool, and then eluting the wiped samples in a phosphate buffer solution or the like for the purpose of confirming viral contamination. The resulting eluate is ultracentrifuged, and the centrifugal sediment is suspended or dissolved and can be used as a specimen (Munemura Yoshiko et al., Food Hygiene Journal, Vol. 58, No. 4, 2017, pp. 201-204).

[0024] Step (3) of the present invention involves mixing the sample with a sample treatment solution containing one or more surfactants. As used herein, "surfactant" is a general term for substances that act on the interface of materials and change their properties. Surfactants have a structure containing both hydrophilic and hydrophobic moieties within the molecule. Surfactants are classified into anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. Anionic surfactants include, but are not limited to, alkyl sulfates, alkyl ether sulfates, docusate, sulfonate fluorosurfactants, alkyl benzene sulfonates, alkyl aryl ether phosphates, alkyl ether phosphates, alkyl carboxylates, sodium lauroyl sarcosinate, carboxylate fluorosurfactants, sodium cholate, and sodium deoxycholate. Preferred alkyl sulfates include sodium dodecyl sulfate (SDS) and ammonium dodecyl sulfate, with sodium dodecyl sulfate being more preferred. Sodium dodecyl sulfate is also known as sodium lauryl sulfate (SLS). Cationic surfactants include, but are not limited to, ethyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, and tetradecyltrimethylammonium bromide. Amphoteric surfactants include, but are not limited to, betaine and alkylamino fatty acid salts. Nonionic surfactants include, but are not limited to, nonylphenoxypolyethoxyethanol (NP-40), polyoxyethylene sorbitan monooleate (Tween® 80), polyoxyethylene pt-octylphenol (Triton X-100®), and the like.

[0025] When surfactants are added to an aqueous solution at a certain concentration or higher, the surfactant monomers aggregate to form micelles. The concentration at which surfactants form micelles is called the critical micelle concentration. In aqueous solution, the hydrophobic regions of proteins and lipids are incorporated into the hydrophobic regions inside the surfactant micelles, solubilizing the proteins and lipids. In RNA virus particles, the protein capsid and lipid envelope are solubilized, denatured, or destroyed in the presence of surfactants at or above the critical micelle concentration. As a result, the RNA encapsulated in the capsid becomes more easily exposed in aqueous solution. The critical micelle concentration of a surfactant varies depending on the type of surfactant. To efficiently expose viral RNA, the surfactant concentration in the sample treatment solution is preferably 0.02 to 0.5% (w / v), more preferably 0.05 to 0.2% (w / v), and even more preferably 0.1% (w / v).

[0026] The mixing ratio of the specimen to the specimen treatment solution is preferably 1:3 to 6 by volume, more preferably 1:4. By mixing the specimen with the specimen treatment solution containing a surfactant, the concentration of the surfactant in the mixture decreases, but the above concentration of the surfactant is such that the critical micelle concentration is maintained.

[0027] In one embodiment of the present invention, the sample treatment solution contains hydroxide. As used herein, "hydroxide" refers to a metal ion as a cation and a hydroxide ion (OH) as an anion. -) is an ionic bond. The metal is an alkali metal or 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 also called alkalis because they are strongly basic and produce hydroxide ions when dissolved in water. In aqueous solution, hydroxides change the charge state of dissociable amino acids such as aspartic acid and glutamic acid in protein molecules, denaturing the proteins. This action causes capsid destruction when RNA virus particles are treated with alkali. As a result, the RNA encapsulated in the capsid is more likely to be exposed in aqueous solution. To efficiently expose the viral RNA, the hydroxide concentration in the sample treatment solution is preferably 10 to 100 mM, more preferably 40 to 60 mM, and even more preferably 50 mM.

[0028] In order to solubilize, denature, or destroy the capsid or lipid envelope and efficiently expose the viral RNA, it is preferable that a surfactant and a hydroxide coexist in the specimen treatment solution.

[0029] Step (3) of the present invention, which efficiently exposes viral RNA from the capsid, is preferably carried out at a temperature of 1 to 60°C, more preferably 1 to 50°C, even more preferably 1 to 40°C, and most preferably at room temperature of 1 to 30°C. After mixing the specimen and specimen treatment solution, it is preferable to leave them to stand for 3 minutes or more. Since step (3) of the present invention does not require heat treatment, there is little risk of bumping or evaporation of the mixture of the specimen and specimen treatment solution in the reaction vessel, and the reaction vessel can be opened without being sealed with a lid or the like.

[0030] Step (4) of the present invention involves mixing the mixture obtained in step (3) with a one-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase, and then performing RT-PCR. In one embodiment of the present invention, step (3) is performed in an unsealed container without a lid. Therefore, steps (3) and (4) can be performed in the same container by directly adding the one-step RT-PCR reaction solution to the container containing the mixture obtained in step (3). Among the surfactants contained in the mixture obtained in step (3), SDS in particular has a strong denaturing effect on proteins. Therefore, if SDS is introduced into step (4) at a high concentration, it may inhibit the enzymatic activity of the reverse transcriptase and DNA polymerase contained in the one-step RT-PCR reaction solution, preventing RT-PCR from proceeding. Similarly, if a high concentration of hydroxide contained in the mixture obtained in step (3) is introduced into step (4), the enzymatic activity may be reduced due to the high pH. Therefore, the mixing ratio of the mixture obtained in step (3) to the one-step RT-PCR reaction solution is preferably 1:2 to 1:6 by volume, more preferably 1:4.

[0031] In step (4) of the present invention, one-step RT-PCR is used to analyze multiple samples in a short time. The one-step RT-PCR reaction mixture contains a reverse transcriptase and a DNA polymerase. Premixed, reverse transcription (single-stranded cDNA synthesis) and PCR can be performed in the same vessel. It can be done.

[0032] The reverse transcriptase contained in the one-step RT-PCR reaction solution is an enzyme that generates single-stranded complementary DNA (cDNA) using viral RNA as a template. There are no particular limitations on the reverse transcriptase as long as it catalyzes the reverse transcription reaction. However, RNA-dependent DNA polymerases derived from RNA viruses such as avian myeloblastosis virus (AMV), Moloney murine leukemia virus (M-MLV), and human immunodeficiency virus (HIV), as well as mutants of these, can be used.

[0033] The DNA polymerase contained in the one-step RT-PCR reaction solution is a thermostable DNA polymerase derived from thermophilic bacteria, and examples include, but are not limited to, Taq, Tth, KOD, Pfu, and their mutants. To avoid nonspecific amplification by the DNA polymerase, a hot-start DNA polymerase may be used. A hot-start DNA polymerase is, for example, a DNA polymerase bound to an anti-DNA polymerase antibody or a DNA polymerase whose enzymatic active site has been chemically modified to be heat-sensitive. In PCR, the DNA polymerase is activated after the initial denaturation step (90°C or higher).

[0034] The one-step RT-PCR reaction solution contains all the components necessary for performing reverse transcription and PCR under appropriate conditions. These components include at least the reverse transcriptase, reverse transcription primer, thermostable DNA polymerase, PCR primer, dNTP mix (a mixture of deoxyribonucleotide 5'-triphosphate; dATP, dGTP, dCTP, and dTTP), and buffer. In one embodiment of the present invention, the reaction solution contains Tris and magnesium. An RNase inhibitor can also be added to the reaction solution. The reverse transcription primer can be a primer specific to the target RNA sequence, an oligo(dT) primer, or a random primer. The PCR primer is a primer pair (forward and reverse) specific to the cDNA sequence generated by the reverse transcription reaction. The PCR primer can be the same as the reverse transcription primer specific to the target RNA sequence. Furthermore, two or more PCR primers can be added to the one-step RT-PCR reaction solution depending on the DNA region to be amplified, i.e., the number of target sequences. As a composition containing the above components, an RT-PCR reaction solution prepared by mixing the reagents contained in a Norovirus Detection Reagent Kit (Probe Method) (Shimadzu Corporation) according to the kit's instruction manual can be used.

[0035] When detecting norovirus RNA, for example, genogroup I (GI) and genogroup II (GII) in norovirus genotypes can be detected by using PCR primers described in Patent Documents 1 and 2, Non-Patent Document 3, and JP 2018-78806, but are not limited thereto. The norovirus detection reagent kit (probe method) includes the PCR primers described in Non-Patent Document 3.

[0036] 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).

[0037] Step (5) of the present invention is a step of detecting the product from the RT-PCR performed in step (4). In one embodiment of the present invention, the PCR product is detected by real-time measurement. When performing the real-time measurement, the RT-PCR in step (4) and the step of detecting the RT-PCR product in step (5) are performed in the same container. In one embodiment of the present invention, step (3) is performed in a non-sealed container without a lid, and therefore steps (3) and (4) can be performed in the same container by directly adding the one-step RT-PCR reaction solution to the container containing the mixture obtained in step (3). Therefore, in one embodiment of the present invention, steps (3) to (5) can be performed in the same container.

[0038] 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 methods using intercalating fluorescent dyes and methods using fluorescently labeled probes. SYBR® Green I is a commonly used intercalating fluorescent dye, but is not limited to this. Intercalating fluorescent dyes bind to double-stranded DNA synthesized by PCR and emit fluorescence when irradiated with excitation light. The amount of PCR amplification product produced can be measured by measuring the intensity of this fluorescence.

[0039] Fluorescently labeled probes include, but are not limited to, TaqMan probes, Molecular Beacons, and cycling probes. TaqMan probes are oligonucleotides modified with a fluorescent dye at the 5' end and a quencher at the 3' end. TaqMan probes specifically hybridize to template DNA during the annealing step of PCR, but the presence of a quencher on the probe suppresses fluorescence emission even when irradiated with excitation light. In the subsequent extension step, the 5'->3' exonuclease activity of Taq DNA polymerase degrades the TaqMan probe hybridized to the template DNA, liberating the fluorescent dye from the probe. This releases the quencher-induced fluorescence suppression, allowing the probe to emit fluorescence. The amount of amplification product produced can be measured by measuring this fluorescence intensity. Examples of fluorescent dyes include, but are not limited to, FAM, ROX, and Cy5. Examples of quenchers include, but are not limited to, TAMRA® and MGB. To distinguish and detect two or more types of DNA target sequences, PCR is performed using two or more types of oligonucleotide probes (for example, TaqMan probes) each bound to a different fluorescent dye.

[0040] In step (5), the amplification curve of the RT-PCR product is measured using a fluorescence filter corresponding to the fluorescent dye used. If the fluorescence intensity increases with the number of PCR cycles, the sample is determined to be positive for the presence of the target RNA virus, while if the fluorescence intensity does not increase during PCR, the sample is determined to be negative.

[0041] In one embodiment of the present invention, a kit for detecting RNA viruses 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. [Example]

[0042] The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples. [Example]

[0043] Effects of detergents and hydroxides on viral RNA exposure in specimens (1) Sample 100 mg of human feces containing norovirus (10 samples) was collected and suspended in 1 mL of distilled water to prepare an approximately 10% (w / v) fecal emulsion. The fecal emulsion was centrifuged at 10,000 rpm for 5 minutes at room temperature, and the supernatant was used as the specimen.

[0044] (2) Sample treatment solution A sample treatment solution containing the following components was prepared. 50 mM sodium hydroxide (NaOH), 0.1% (w / v) sodium dodecyl sulfate (SDS), and 625 μM dNTPs (dATP, dGTP, dCTP, and dTTP)

[0045] (3-1) Sample processing 4 μL of the sample treatment solution was placed in a PCR reaction tube without a lid, and 1 μL of the sample was added thereto, followed by leaving it at room temperature for 3 minutes. (3-2) Sample processing involving heat treatment For comparison, a sample was treated with a sample treatment solution containing 15 mM NaOH but no SDS. The sample treatment solution used was the same as that included in the Norovirus Detection Reagent Kit (Probe Method) (Shimadzu Corporation, Product Number 241-09325-91). 9 μL of the sample treatment solution was placed in a PCR reaction tube, and 1 μL of the sample was added thereto and stirred. The tube was then spun down in a small centrifuge, then placed in a thermostatic chamber at 90°C, and heat-treated for 5 minutes. After this heat treatment, the PCR reaction tube was spun down in a small centrifuge and cooled on ice.

[0046] (4) One-step RT-PCR reaction 20 μL of the 1-step RT-PCR reaction mixture was added to a PCR reaction tube containing 5 μL of the treatment solution obtained in 3-1 above, or 15 μL of the 1-step RT-PCR reaction mixture was added to a PCR reaction tube containing 10 μL of the treatment solution obtained in 3-2 above, and the mixture was stirred and mixed, then spun down in a small centrifuge. After this, the reaction was immediately started using a real-time PCR device (GVP-9600, Shimadzu Corporation).

[0047] (5) Composition of one-step RT-PCR reaction solution To 5 μL of the treatment solution obtained in 3-1 above, a one-step RT-PCR reaction solution was added so that the reaction solution composition was as follows: To 10 μL of the treatment solution obtained in 3-2 above, a one-step RT-PCR reaction solution prepared by mixing the reagents (NoV Reagents A, B, and C) included in the Norovirus Detection Reagent Kit (probe method) (Shimadzu Corporation, product number 241-09325-91) was added. The composition during the reaction was as follows: 40mM Tris 0.025 units / μL Taq polymerase 1 unit / μL reverse transcriptase 3.75mM magnesium chloride 400 nM PCR primer set (COG1F / COG1R and COG2F / COG2R) (see Non-Patent Document 3, Table 11) 200 nM fluorescently labeled probes (TaqMan probes: G1A, G1B, and G2)

[0048] (6) RT-PCR setting conditions After reverse transcription at 45°C for 5 minutes, initial denaturation was performed at 95°C for 3 minutes, followed by 45 cycles of PCR at 95°C for 1 second and 56°C for 10 seconds. Photometry was performed at the 56°C / 10 second step.

[0049] (7) Results and Discussion The photometric results are shown in Table 1. Table 1 compares the Ct values ​​obtained when samples were treated with the sample treatment solution of the present invention and when samples were treated with conventional heat treatment. The Ct value is the cycle number at which the amplification curve intersects with the threshold in real-time PCR. Table 1 shows that the Ct values ​​for all samples were nearly identical between the conventional heat treatment and the treatment with the sample treatment solution of the present invention. This result indicates that the initial template amount was nearly identical in both treatments. In other words, the sample treatment of the present invention, which can eliminate heat treatment by using a surfactant and hydroxide, has the same viral RNA exposure effect as the conventional heat treatment method.

[0050] [Table 1]

Claims

1. 1. A method for detecting an RNA virus in a sample, comprising: (1) suspending a specimen in distilled water, physiological saline, or a buffer solution; (2) extracting the supernatant from the suspension produced in step (1); (3) mixing the centrifugal supernatant extracted in step (2) with a sample treatment solution containing hydroxides consisting of one or both of sodium hydroxide and potassium hydroxide and one or more surfactants to obtain a mixed solution; (4) mixing the mixture obtained in step (3) with a one-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase, and performing RT-PCR; and (5) detecting the RT-PCR product; Including, the one-step RT-PCR reaction solution contains at least Tris; the hydroxide concentration in the specimen treatment solution is 10 to 100 mM; the concentration of the surfactant in the specimen treatment solution is 0.02 to 0.5% (w / v); The method, wherein the solution obtained by mixing the mixture with the one-step RT-PCR reaction solution is alkaline.

2. 2. The method of 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.

3. The method of claim 1 , wherein the RNA virus is a norovirus.

4. The method of claim 3, wherein the genotype of the Norovirus is genogroup I (GI) or genogroup II (GII).

5. 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 biological sample, a biologically derived sample, an environmental sample, and an environmentally derived sample.

6. The method of any one of claims 1 to 4, wherein the specimen is derived from a sample selected from the group consisting of a fecal sample, a fecal-derived sample, vomit, and a vomit-derived sample.

7. The method according to any one of claims 1 to 6, wherein the surfactant is an anionic surfactant.

8. 8. The method of claim 7, wherein the anionic surfactant is one or more anionic surfactants selected from the group consisting of alkyl sulfates, alkyl ether sulfates, docusate, sulfonate fluorosurfactants, alkyl benzene sulfonates, alkyl aryl ether phosphates, alkyl ether phosphates, alkyl carboxylates, sodium lauroyl sarcosinate, carboxylate fluorosurfactants, sodium cholate, and sodium deoxycholate.

9. The method of claim 7 wherein the anionic surfactant is an alkyl sulfate.

10. 10. The method of claim 9, wherein the alkyl sulfate is sodium dodecyl sulfate or ammonium dodecyl sulfate.

11. The method according to any one of claims 1 to 10, wherein the mixing ratio of the specimen to the specimen treatment solution in step (3) is 1:3 to 1:6 by volume.

12. The method according to any one of claims 1 to 11, wherein the reverse transcriptase is selected from the group consisting of AMV reverse transcriptase, MMLV reverse transcriptase, HIV reverse transcriptase, and mutants thereof.

13. The method according to any one of claims 1 to 12, wherein the DNA polymerase is selected from the group consisting of Taq DNA polymerase, Tth DNA polymerase, KOD DNA polymerase, Pfu DNA polymerase, and mutants thereof.

14. The method according to any one of claims 1 to 13, wherein the step (3) is carried out at a temperature of 1 to 60°C.

15. The method according to any one of claims 1 to 14, wherein steps (3) to (5) are carried out in the same container.

16. The method according to any one of claims 1 to 15, wherein in the step (5), an amplification curve of the RT-PCR product is measured using a fluorescent filter, and the presence of an RNA virus in the sample is determined to be positive or negative.

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