CN114080456A - RNA virus detection method - Google Patents

RNA virus detection method Download PDF

Info

Publication number
CN114080456A
CN114080456A CN202080049541.7A CN202080049541A CN114080456A CN 114080456 A CN114080456 A CN 114080456A CN 202080049541 A CN202080049541 A CN 202080049541A CN 114080456 A CN114080456 A CN 114080456A
Authority
CN
China
Prior art keywords
sample
virus
kit
pcr
norovirus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202080049541.7A
Other languages
Chinese (zh)
Inventor
小林慎一郎
四方正光
高冈直子
二宫健二
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shimadzu Corp
Original Assignee
Shimadzu Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shimadzu Corp filed Critical Shimadzu Corp
Publication of CN114080456A publication Critical patent/CN114080456A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention relates to a method for detecting RNA viruses by reverse transcription-polymerase chain reaction (RT-PCR), and a kit for performing the method. And more particularly to: a method for detecting an RNA virus by mixing a sample with a surfactant in the presence of a hydroxide and further adding an RT-PCR reaction solution; and a kit for performing the method.

Description

RNA virus detection method
Technical Field
The present invention relates to a method for detecting RNA viruses by reverse transcription-polymerase chain reaction (RT-PCR), and a kit for performing the method. And more particularly to: a method for detecting an RNA virus by mixing a sample with a surfactant in the presence of a hydroxide and further adding an RT-PCR reaction solution; and a kit for performing the method.
Background
RNA viruses are viruses having RNA as a genome, and are classified into coronavirus, human immunodeficiency virus, hepatitis c virus, japanese encephalitis virus, dengue virus, and the like, which have an envelope that is a membrane composed of a lipid bilayer; and norovirus, rotavirus, rhinovirus, etc. without envelope, and have many pathogenicity.
Norovirus is a virus belonging to the family human caliciviridae, and has a single-stranded RNA of about 7000 bases in the genome. This Virus is also known as Small Round Virus (SRSV) according to the morphological classification observed by electron microscopy and is a Virus under the generic name Norwalk-like Virus. Viruses belonging to norovirus are classified into 2 genomes, genome (genoreup) i (gi) and genome ii (gii), and further into 14 and 17, or more genotypes (genotypes), respectively.
If a person infects norovirus, acute gastroenteritis symptoms such as vomiting and diarrhea appear. About half of the food poisoning patients in japan are caused by norovirus each year, about 70% of which occurs in 11 to 2 months, and norovirus is known to be a causative virus of winter-type gastroenteritis and food poisoning. Food poisoning by norovirus is mainly caused by contamination of food by chefs. Norovirus has strong infectivity and is easy to cause epidemic situations such as large-scale food poisoning and the like. The route of transmission to humans is primarily oral infection. Typical sources of infection include feces and vomit of infected persons, and products directly or indirectly contaminated with these, and foods such as oysters or other bivalve shellfish contaminated with norovirus. Therefore, the identification of norovirus-infected patients, contaminants contaminated by the virus, is particularly important to prevent the spread of viral infections.
As virus detection for detecting infection or contamination by a virus, an immunoassay method for detecting a virus antigen or a virus gene amplification method (patent documents 1 to 3 and non-patent document 1) can be used. As a means for measuring norovirus with high sensitivity, a method of amplifying RNA of norovirus by RT-PCR and measuring the amount of the amplified product can be mentioned. For example, detection of norovirus by the RT-PCR method and quantitative detection of norovirus by the real-time PCR method have been widely carried out in response to the notice of monitoring of safety lessons by the food safety department of the ministry of health and labor, japan (non-patent documents 2 and 3).
RNA virions have the following basic structure: a core consisting of an RNA genome and proteins is enclosed within a protein shell called a capsid. Therefore, in order to detect viral RNA by a gene amplification method, RNA needs to be extracted from viral particles. In order to detect norovirus in feces as a sample, for example, the feces sample is suspended in distilled water or physiological saline at a concentration of 5 to 10% (w/v), and from the supernatant, RNA is extracted and purified using a commercially available Viral RNA extraction kit (for example, QIAamp (registered trademark) Viral RNA Mini, QIAGEN corporation) (non-patent document 2). However, it is complicated to perform the RT-PCR detection process after performing the RNA extraction and purification operations in a plurality of stages. Therefore, the following simple assay is proposed: the fecal suspension and the sample treatment solution are mixed, and heat-treated for a short time to remove the coat protein, thereby liberating the internal RNA, and the liberated RNA is directly subjected to RT-PCR (non-patent document 4). On the other hand, in order to heat-treat a mixture of the fecal suspension and the treatment solution for specimen, it is necessary to seal the reaction vessel with a cap to prevent bumping and evaporation of the mixture, and to add the RT-PCR reaction solution after removing the cap after the heat treatment. To improve this, the following methods are proposed: a test sample is mixed with a chaotropic agent such as a guanidine salt to detect a virus by RT-PCR without heat treatment (patent document 4).
Documents of the prior art
Patent document
Patent document 1: WO2002/029119
Patent document 2: WO2002/029120
Patent document 3: japanese laid-open patent publication No. 2004-301684
Patent document 4: japanese patent laid-open publication No. 2017-209036
Non-patent document
Non-patent document 1: kageyama T, et al, Broadly reactive and highlyls reactive assay 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: the food safety department of the japan ministry of health, labor, and industry for medicine and food administration for monitoring safety class food safety administration, No. 1105001 (heicheng 15, 11, 5 days)' ノロウイルス zhi quan u shi について (detection method for norovirus) ", and finally revised: food safety supervision No. 0514004 (Pingcheng 19 years, 5 months and 14 days)
Non-patent document 3: the food safety department of the japan ministry of health, labor, and industry for medicine and food administration for monitoring safety class food safety administration, No. 1105001 (heicheng 15, 11, 5 days)' ノロウイルス zhi quan u shi について (detection method for norovirus) ", and finally revised: food safety administration 1022 No. 1 (Pingcheng 25 years 10 months 22 days)
Non-patent document 4: detection of microorganisms in functional specialimens by direct RT-PCR with RNA purification. J Virol methods.2010 Feb; 163(2):282-286.
Disclosure of Invention
Problems to be solved by the invention
The purpose of the present invention is to provide a simple method for detecting an RNA virus. Specifically, the following method is provided: RNA is extracted from RNA viral particles such as norovirus using 1 or more surfactants without heat treatment, and then virus detection is easily performed by RT-PCR reaction using free RNA. In addition, the following detection methods are provided: a method for detecting RNA viruses in a simpler manner by extracting RNA from RNA viral particles and performing RT-PCR in the same vessel without opening and closing the lid of the vessel.
Means for solving the problems
The object of the present invention is achieved by the following invention.
〔1〕
A method for detecting an RNA virus in a sample, comprising the steps of:
(1) suspending the sample in distilled water, physiological saline or a buffer solution;
(2) a step of extracting a supernatant of the suspension produced in the step (1) by centrifugation;
(3) mixing the centrifugal supernatant extracted in the step (2) with a sample treatment solution containing 1 or more kinds of surfactants;
(4) a step of mixing the mixed solution obtained in the step (3) with a one-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase to perform RT-PCR; and
(5) and (3) detecting the RT-PCR product.
〔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 a norovirus.
〔4〕
The method according to [ 3 ], wherein the norovirus genotype is Genome I (GI) or genome 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 a biological sample, a sample derived from a biological source, an environmental sample and a sample derived from an environmental source.
〔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 an excrement sample, an excrement-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.
〔8〕
The method according to [ 7 ], wherein the anionic surfactant is at least 1 anionic surfactant selected from the group consisting of alkyl sulfate, alkyl ether sulfate, docusate (docusate), sulfonate fluorosurfactant, alkylbenzene sulfonate, alkylaryl ether phosphate, alkyl carboxylate, sodium lauroyl sarcosinate, carboxylate fluorosurfactant, sodium cholate and sodium deoxycholate.
〔9〕
The process according to [ 7 ], wherein the anionic surfactant is an alkyl sulfate.
〔10〕
The process according to [ 9 ], wherein the alkyl sulfate is sodium lauryl sulfate or ammonium lauryl 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 process according to [ 12 ], wherein the hydroxide is sodium hydroxide or potassium hydroxide.
〔14〕
The method according to [ 12 ] or [ 13 ], wherein the hydroxide is used in a concentration of 10 to 100 mM.
〔15〕
The method according to any one of [ 1] to [ 14 ], wherein a mixing ratio of the centrifugal supernatant liquid and the sample treatment liquid in the step (3) is 1: 3 to 6.
〔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 a mutant thereof.
〔18〕
The method according to any one of [ 1] to [ 17 ], wherein the step (5) is performed by real-time measurement.
〔19〕
The method according to any one of [ 1] to [ 18 ], wherein the step (3) is performed at a temperature of 1 to 60 ℃.
〔20〕
The method according to any one of [ 1] to [ 19 ], wherein the steps (3) to (5) are carried out in the same vessel.
〔21〕
The method according to any one of [ 1] to [ 20 ], wherein in the step (5), the presence of the RNA virus in the specimen is determined to be positive or negative by measuring an amplification curve of the RT-PCR product using a fluorescence filter.
〔22〕
A kit for the detection of an RNA virus comprising: a sample treatment solution containing 1 or more kinds of surfactants, and a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase.
〔23〕
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.
〔24〕
The kit according to [ 22 ], wherein the RNA virus is a norovirus.
〔25〕
The kit according to [ 24 ], wherein the genotype of the norovirus is judged as either Genome I (GI) or genome II (GII).
〔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 at least 1 anionic surfactant selected from the group consisting of alkyl sulfate, alkyl ether sulfate, docusate, sulfonate fluorosurfactant, alkylbenzene sulfonate, alkylaryl ether phosphate, alkyl carboxylate, sodium lauroyl sarcosinate, carboxylate fluorosurfactant, 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 lauryl sulfate or ammonium lauryl sulfate.
〔30〕
The kit according to any one of [ 22 ] to [ 29 ], which further comprises instructions for the procedures of the kit.
ADVANTAGEOUS EFFECTS OF INVENTION
According to the present invention, by mixing a centrifuged supernatant of a sample suspension containing RNA viral particles such as norovirus with a sample treatment solution containing 1 or more kinds of surfactants, RNA can be efficiently released from the viral particles without heat treatment. Therefore, a series of operations including the treatment of liberating RNA and the subsequent addition of RT-PCR reaction solution for detecting the presence of virus can be continuously performed in the same vessel, and RNA virus can be easily detected. Further, in the present invention, since the efficiency of liberating RNA from virus particles is high, the virus detection sensitivity is high, and the virus excretion period can be accurately detected. And is therefore useful for detecting occult infections, especially for determining infected patients in the convalescent phase after infection.
Detailed Description
The present invention provides a method for detecting an RNA virus in a sample. The method comprises the following steps: (1) suspending the sample in distilled water, physiological saline or a buffer solution; (2) a step of extracting a supernatant of the suspension produced in the step (1) by centrifugation; (3) mixing the centrifugal supernatant extracted in the step (2) with a sample treatment solution containing 1 or more kinds of surfactants; (4) a step of mixing the mixed solution obtained in the step (3) with a one-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase to perform RT-PCR; and (5) detecting the RT-PCR product.
In the present invention, the RNA virus to be detected in the sample is a virus having RNA as a genome, and examples thereof include coronavirus having an envelope which is a membrane composed of a lipid bilayer, human immunodeficiency virus, hepatitis C virus, Japanese encephalitis virus, and dengue virus; and norovirus, rotavirus, rhinovirus, etc. without envelope, but are not limited thereto. Since the envelope is mainly composed of lipid, it is easily broken by an organic solvent such as alcohol or a surfactant, but RNA viruses such as norovirus that do not have such an envelope generally exhibit resistance to organic solvents or surfactants.
Examples of the sample in the present invention include biological samples, environmental samples, and environmental samples. The biological sample includes animal and plant tissues including the midgut gland of shellfish, and body fluids such as blood, saliva, nasal discharge, and tissue secretion. In particular, shellfish has been attracting attention as a food product causing food poisoning by norovirus. Examples of the biological sample include those obtained by subjecting the biological sample to a treatment such as an ultrasonic treatment. Examples of the environmental sample include all samples including air, soil, dust, water, and the like. Examples of the environmental source sample include those obtained by subjecting the environmental sample to a treatment such as an ultrasonic treatment.
In another embodiment of the present invention, the sample includes an excrement sample, an excrement-derived sample, vomit, a vomit-derived sample, and the like. The fecal sample and the vomit sample may be used as they are, or may be suspended in distilled water, physiological saline or a buffer solution at a concentration of, for example, 10% (w/v) to prepare an emulsion as the step (1). The buffer is not particularly limited, and examples thereof include Good's buffers such as phosphate buffer, Tris buffer, borate buffer, and HEPES buffer. The emulsion of the sample may be centrifuged at 10000 to 12000rpm for 2 to 20 minutes to remove impurities such as bacteria in the intestinal tract, and the centrifuged supernatant may be used as a sample as a step (2). The fecal and vomit source samples include swab samples. The wiping sample is obtained by wiping fingers, tableware, chopping boards, kitchen knives, cookers, toilet facilities, housing facilities, etc. with a cotton swab, a cotton ball, etc. and then eluting the wiped sample in a phosphate buffer solution, etc. in order to confirm viral contamination. The obtained eluate can be subjected to ultracentrifugation, and the centrifuged precipitate can be suspended or dissolved and used as a sample (p.201-204 No. 4, volume 58, 2017, journal of food hygiene, e.g., chazu junzi).
Step (3) of the present invention is a step of mixing a sample with a sample treatment solution containing 1 or more kinds of surfactants. In the present specification, the term "surfactant" refers to a general term for a substance that acts on a boundary surface of a substance to change its properties. The surfactant has a structure containing both a hydrophilic portion and a hydrophobic portion in a molecule. The surfactant is classified into an anionic surfactant, a cationic surfactant, an amphoteric surfactant and a nonionic surfactant. Examples of the anionic surfactant include, but are not limited to, alkyl sulfates, alkyl ether sulfates, docusates, sulfonate fluorosurfactants, alkylbenzene sulfonates, alkylaryl ether phosphates, alkyl carboxylates, sodium lauroyl sarcosinate, carboxylate fluorosurfactants, 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 Lauryl Sulfate is also known as Sodium Lauryl Sulfate (SLS). Examples of the cationic surfactant include, but are not limited to, ethyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, and tetradecyltrimethylammonium bromide. Examples of the amphoteric surfactant include, but are not limited to, betaines and alkyl amino fatty acid salts. Examples of the nonionic surfactant include, but are not limited to, nonylphenoxypolyethoxyethanol (NP-40), polyoxyethylene sorbitan monooleate (Tween (registered trademark) 80), and polyoxyethylene p-tert-octylphenol (Triton X-100 (registered trademark)).
When the surfactant is added to an aqueous solution at a concentration of at least a certain level, surfactant monomers aggregate to form micelles. The concentration at which the surfactant will form micelles is referred to as the critical micelle concentration. In an aqueous solution, hydrophobic regions of proteins and lipids enter hydrophobic regions inside surfactant micelles, and the proteins and lipids are solubilized. In RNA viral particles, a capsid serving as a protein shell or an envelope composed of a lipid is solubilized, denatured, or destroyed in the presence of a surfactant having a critical micelle concentration or higher. As a result, the RNA encapsulated in the capsid is easily exposed to an aqueous solution. The critical micelle concentration of the surfactant varies depending on the type of the surfactant, and the concentration of the surfactant in the sample treatment solution is preferably 0.02 to 0.5% (w/v), more preferably 0.05 to 0.2% (w/v), and still more preferably 0.1% (w/v) in order to efficiently expose viral RNA.
The mixing ratio of the sample to be tested and the sample treatment solution is preferably 1: 3-6, more preferably 1: 4. by mixing the sample with the sample treatment solution containing the surfactant, the concentration of the surfactant in the mixed solution is reduced, but the above concentration of the surfactant maintains the critical micelle concentration.
In one embodiment of the present invention, the sample processing liquid contains a hydroxide. In the present specification, "hydroxide" refers to a substance in which a metal ion as a cation is ionically bonded to a hydroxide ion (OH-) as an anion. The metal is an alkali metal or an alkaline earth metal. As the hydroxide, lithium hydroxide, sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide, and barium hydroxide can be exemplified, with sodium hydroxide and potassium hydroxide being preferred. Hydroxides exhibit strong basicity and generate hydroxide ions when dissolved in water and are therefore also referred to as bases. The hydroxide changes the charge state of a dissociative amino acid such as aspartic acid or glutamic acid in a protein molecule in an aqueous solution, thereby denaturing the protein. By this action, capsid destruction occurs when the RNA virions are subjected to alkaline treatment. As a result, the RNA encapsulated in the capsid is easily exposed to an aqueous solution. In order to efficiently expose viral RNA, the hydroxide concentration in the sample treatment solution is preferably 10 to 100mM, more preferably 40 to 60mM, and still more preferably 50 mM.
In order to solubilize, denature or destroy the capsid and the envelope composed of lipid to efficiently expose the viral RNA, it is preferable to allow a surfactant and a hydroxide to coexist in the treatment solution for the specimen.
The step (3) of the present invention for efficiently exposing viral RNA to capsid is preferably carried out at a temperature of 1 to 60 ℃, more preferably at 1 to 50 ℃, even more preferably at 1 to 40 ℃, and most preferably at room temperature of 1 to 30 ℃. Preferably, the sample is mixed with the sample treatment solution and then left for 3 minutes or more. Since the step (3) of the present invention does not require heat treatment, the mixed solution of the sample and the sample treatment liquid in the reaction vessel is less likely to boil or evaporate, and can be carried out in an open state without closing the reaction vessel with a lid or the like.
Step (4) of the present invention is a step of mixing the mixed solution obtained in step (3) with a one-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase and performing RT-PCR. In one embodiment of the present invention, since the step (3) is performed in a non-sealed container without using a lid, the 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 mixed solution obtained in the step (3) in this state. Among the surfactants contained in the mixed solution obtained in step (3), SDS has a particularly strong protein denaturing effect. Therefore, when SDS is introduced at a high concentration in the step (4), the enzyme activities of the reverse transcriptase and DNA polymerase contained in the one-step RT-PCR reaction solution are inhibited, and there is a possibility that RT-PCR cannot be performed. Similarly, when the concentration of the hydroxide contained in the mixed solution obtained in step (3) is high, the enzyme activity is decreased by a high pH, even when the concentration is high in step (4). Therefore, the mixing ratio of the mixed solution obtained in the step (3) to the one-step RT-PCR reaction solution is preferably 1: 2-6, more preferably 1: 4.
in step (4) of the present invention, one-step RT-PCR is used 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-stranded cDNA synthesis) and PCR can be performed in the same vessel.
The reverse transcriptase contained in the one-step RT-PCR reaction solution is not particularly limited as long as it is an enzyme that generates a single-stranded complementary DNA (cDNA) using viral RNA as a template, and 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) and mutants thereof can be used as long as they catalyze the reverse transcription reaction.
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 their mutants can be used, but the DNA polymerase is not limited thereto. In order to avoid non-specific amplification by the DNA polymerase, a hot start DNA polymerase may also be used. 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 active site of the enzyme is subjected to a heat-sensitive chemical modification, and is an enzyme which is activated only after an initial denaturation step (at 90 ℃ or higher) in PCR.
The one-step RT-PCR reaction solution contains all the components for smoothly performing the reverse transcription reaction and the PCR under appropriate conditions. The composition at least comprises: the reverse transcriptase, the reverse transcription primer, the thermostable DNA polymerase, the PCR primer, a dNTP mixture (deoxyribonucleoside-5' -triphosphate; a mixture of dATP, dGTP, dCTP and dTTP), and a buffer. In one embodiment of the present invention, the reaction solution contains a Tris buffer and magnesium. An RNase inhibitor may be added to the reaction solution. As the reverse transcription primer, a primer specific for a target RNA sequence, oligo (dT) primer or random primer can be used. As the PCR primer, a primer pair (forward and reverse) specific to the sequence of cDNA generated by a reverse transcription reaction can be used. The PCR primers may be the same as the aforementioned reverse transcription primers specific for the target RNA sequence. In addition, 2 or more PCR primers may be added to the one-step RT-PCR reaction solution depending on the DNA region to be amplified, that is, the number of target sequences. As the composition containing the above-mentioned components, an RT-PCR reaction solution obtained by mixing reagents contained in a norovirus detection kit (probe method) (Shimadzu corporation) according to the kit protocol can be used.
In the detection of norovirus RNA, for example, the Genome I (GI) and the genome II (GII) in the norovirus genotype can be detected by using PCR primers described in patent documents 1 and 2, non-patent document 3, and Japanese patent application laid-open No. 2018-78806, but the invention is not limited thereto. The norovirus detection kit (probe method) includes a PCR primer described in non-patent document 3.
The reaction temperature conditions for the reverse transcription reaction in RT-PCR, and the PCR conditions (temperature, time, and number of cycles) can be easily set by those skilled in the art.
Step (5) of the present invention is a step of detecting a product derived from RT-PCR performed in step (4). In one embodiment of the invention, the PCR product is detected by real-time assay. In the real-time measurement, the RT-PCR in the step (4) and the step of detecting the RT-PCR product in the step (5) are carried out in the same vessel. In one embodiment of the present invention, since the step (3) is performed in a non-sealed container without using a lid, the 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 mixed solution obtained in the step (3) in this state. Therefore, in one embodiment of the present invention, the steps (3) to (5) can be performed in the same container.
Real-time determination of PCR products is also referred to as real-time PCR. In real-time PCR, PCR amplification products are typically detected by fluorescence. The fluorescence detection method comprises the following steps: a method using an intercalating fluorescent dye and a method using a fluorescently labeled probe. As the intercalating fluorescent dye, SYBR (registered trademark) Green I can be used, but 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 the fluorescence intensity, the amount of the PCR amplification product produced can be measured.
Examples of the fluorescent-labeled probe include, but are not limited to, a TaqMan probe, a Molecular Beacon (Molecular Beacon), and a circular probe. The TaqMan probe is an oligonucleotide having a 5 'end modified with a fluorescent dye and a 3' end modified with a quencher substance. The TaqMan probe specifically hybridizes to the template DNA in the annealing step of PCR, but the generation of fluorescence is suppressed even when excitation light is irradiated thereto because of the presence of a quencher on the probe. In the subsequent extension reaction step, when the TaqMan probe hybridized with the template DNA is degraded by the 5 '→ 3' exonuclease activity of Taq DNA polymerase, the fluorescent dye is released from the probe, and the quencher suppresses the generation of fluorescence, thereby emitting fluorescence. By measuring the fluorescence intensity, the amount of the amplification product produced can be measured. Examples of the fluorescent dye include FAM, ROX, and Cy5, but are not limited thereto. Examples of the quencher include TAMRA (registered trademark) and MGB, but are not limited thereto. In order to detect 2 or more DNA target sequences separately, PCR is performed using 2 or more oligonucleotide probes (e.g., TaqMan probes) to which different fluorescent dyes are bound.
In the step (5), the amplification curve of the RT-PCR product is measured using a fluorescence filter corresponding to the fluorescent dye used. When the fluorescence intensity increases according to the number of cycles of PCR, the presence of RNA virus to be analyzed in the specimen is judged to be positive, and when the fluorescence intensity does not increase in PCR, the presence of RNA virus to be analyzed in the specimen is judged to be negative.
In one embodiment of the present invention, there is provided a kit for detecting an RNA virus, comprising: a sample treatment solution containing 1 or more kinds of surfactants, and a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase.
Examples
The present invention will be described in detail with reference to examples, but the present invention is not limited thereto.
Example 1
[ Effect of the surfactant and hydroxide on the Exposure of viral RNA to the specimen ]
(1) Sample to be tested
100mg of human feces (10 cases) containing norovirus were collected and suspended in 1mL of distilled water to prepare about 10% (w/v) feces emulsion. The feces emulsion was centrifuged at 10000rpm for 5 minutes at room temperature, and the centrifuged supernatant was used as a sample.
(2) Liquid for treating specimen
A liquid to be treated containing the following components was prepared.
50mM sodium hydroxide (NaOH),
0.1% (w/v) Sodium Dodecyl Sulfate (SDS), and
625 μ M dNTP (dATP, dGTP, dCTP and dTTP)
(3-1) treatment of the specimen
mu.L of the sample treatment solution was taken out into a PCR reaction tube without a cap, and 1. mu.L of the sample was added thereto, followed by standing at room temperature for 3 minutes.
(3-2) treatment of specimen with Heat treatment
For comparison, a sample was treated with a sample treatment solution containing 15mM NaOH but not SDS. As the Sample Treatment solution, a Sample Treatment solution (Sample Treatment Reagent) contained in a norovirus detection kit (Probe method) (Shimadzu corporation, product No. 241-09325-91) was used. After 9. mu.L of the treatment solution for a specimen was taken out into a PCR reaction tube, 1. mu.L of the specimen was put into the reaction tube, and mixed with stirring, and centrifuged briefly (spin down) in a small centrifugal separator, the reaction tube was placed in a thermostatic apparatus at 90 ℃ and heat-treated for 5 minutes. After this heat treatment, the PCR reaction tube was centrifuged briefly by a small centrifugal separator and directly cooled with ice.
(4) One-step RT-PCR reaction
Adding 20. mu.L of one-step RT-PCR reaction solution to a PCR reaction tube containing 5. mu.L of the treatment solution obtained in 3-1 above; alternatively, 15. mu.L of the one-step RT-PCR reaction solution was added to a PCR reaction tube containing 10. mu.L of the treated solution obtained in 3-2, and the mixture was stirred and mixed, followed by brief centrifugation in a small centrifugal separator. Then, the reaction was immediately started by using a real-time PCR apparatus (GVP-9600, Shimadzu corporation).
(5) Composition of one-step RT-PCR reaction solution
To 5. mu.L of the treated solution obtained in the above 3-1, a one-step RT-PCR reaction solution was added so as to have the following reaction composition. To 10. mu.L of the treated solution obtained in the above 3-2 was added a one-step RT-PCR reaction solution prepared by mixing the reagents (NoV Reagent A, B and C) contained in the norovirus detection kit (Probe method) (Shimadzu Corp., product No. 241-09325-91). The composition in the reaction is as follows.
40mM Tris buffer
0.025 units/. mu.L Taq polymerase
1 Unit/. mu.L reverse transcriptase
3.75mM magnesium chloride
400nM PCR primer sets (COG1F/COG1R and COG2F/COG2R) (see non-patent document 3, Table 11)
200nM fluorescently labeled probe (TaqMan probe: G1A, G1B and G2)
(6) Setting conditions for RT-PCR
After a "45 ℃/5 min" reverse transcription reaction, an initial denaturation of "95 ℃/3 min" was performed followed by 45 cycles of PCR of "95 ℃/1 sec-56 ℃/10 sec". The optical measurements were performed in 56 deg.C/10 sec steps.
(7) Results and investigation
The optical measurement results are shown in table 1. Table 1 is a graph comparing the case of treating a specimen with the specimen treatment liquid of the present invention and the case of performing heat treatment, which is a conventional method, and shows Ct values. The Ct value is the number of cycles that the amplification curve crosses a Threshold (Threshold) in real-time PCR. Table 1 shows: in the case of the conventional method, that is, the heat treatment, and the case of the treatment with the sample treatment solution of the present invention, Ct values are almost the same for all samples to be examined. The results show that: in any of the processes, the initial template amount is almost the same. Namely, it can be seen that: the treatment of the specimen of the present invention, which can omit the heat treatment by using a surfactant and a hydroxide, has a virus RNA exposure effect equivalent to that of the conventional method in which heat treatment is performed.
[ Table 1]
Figure BDA0003458658230000161

Claims (30)

1. A method for detecting an RNA virus in a sample, comprising the steps of:
(1) suspending the sample in distilled water, physiological saline or a buffer solution;
(2) a step of extracting a supernatant of the suspension produced in the step (1) by centrifugation;
(3) mixing the centrifugal supernatant extracted in the step (2) with a sample treatment solution containing 1 or more kinds of surfactants;
(4) a step of mixing the mixed solution obtained in the step (3) with a one-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase to perform RT-PCR; and
(5) and detecting the RT-PCR product.
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 norovirus genotype is Genome I (GI) or genome II (GII).
5. The method according to any one of claims 1 to 4, wherein the sample is derived from a sample selected from the group consisting of a biological sample, a sample derived from a biological source, an environmental sample and a sample derived from an environmental source.
6. The method according to any one of claims 1 to 4, wherein the test sample is derived from a sample selected from the group consisting of an excrement sample, an excrement-derived sample, vomit and a vomit-derived sample.
7. A process according to any one of claims 1 to 6, wherein the surfactant is an anionic surfactant.
8. The method of claim 7, wherein the anionic surfactant is 1 or more anionic surfactants selected from the group consisting of alkyl sulfates, alkyl ether sulfates, docusates, sulfonate fluorosurfactants, alkylbenzene sulfonates, alkylaryl 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. The method of claim 9, wherein the alkyl sulfate is sodium or ammonium lauryl sulfate.
11. 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).
12. 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. The method according to claim 12 or 13, wherein the hydroxide is present in a concentration of 10 to 100 mM.
15. The method according to any one of claims 1 to 14, wherein the mixing ratio of the sample to the sample treatment solution in the step (3) is 1: 3 to 6.
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 mutants 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 mutants thereof.
18. The method according to any one of claims 1 to 17, wherein the step (5) is performed by real-time measurement.
19. The method according to any one of claims 1 to 18, wherein the step (3) is carried out at a temperature of 1 to 60 ℃.
20. The method according to any one of claims 1 to 19, wherein the steps (3) to (5) are carried out in the same vessel.
21. The method according to any one of claims 1 to 20, wherein in the step (5), the presence of RNA virus in the sample is determined to be positive or negative by measuring the amplification curve of the RT-PCR product using a fluorescence filter.
22. A kit for the detection of an RNA virus comprising: a sample treatment solution containing 1 or more kinds of surfactants, and a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase.
23. The kit according to claim 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.
24. The kit of claim 22, wherein the RNA virus is a norovirus.
25. The kit according to claim 24, wherein the norovirus genotype is judged to be genome i (gi) or genome ii (gii).
26. The kit according to any one of claims 22 to 25, wherein the surfactant is an anionic surfactant.
27. The kit according to claim 26, wherein the anionic surfactant is 1 or more anionic surfactants selected from the group consisting of alkyl sulfate, alkyl ether sulfate, docusate, sulfonate fluorosurfactant, alkylbenzene sulfonate, alkylaryl ether phosphate, alkyl carboxylate, sodium lauroyl sarcosinate, carboxylate fluorosurfactant, sodium cholate and sodium deoxycholate.
28. The kit of claim 26, wherein the anionic surfactant is an alkyl sulfate.
29. The kit of claim 28, wherein the alkyl sulfate is sodium or ammonium lauryl sulfate.
30. The kit of any one of claims 22 to 29, further comprising instructions for the procedures of the kit.
CN202080049541.7A 2019-07-18 2020-07-08 RNA virus detection method Pending CN114080456A (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2019-133045 2019-07-18
JP2019133045 2019-07-18
PCT/JP2020/026646 WO2021010239A1 (en) 2019-07-18 2020-07-08 Rna virus detection method

Publications (1)

Publication Number Publication Date
CN114080456A true CN114080456A (en) 2022-02-22

Family

ID=74210664

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202080049541.7A Pending CN114080456A (en) 2019-07-18 2020-07-08 RNA virus detection method

Country Status (3)

Country Link
JP (2) JPWO2021010239A1 (en)
CN (1) CN114080456A (en)
WO (1) WO2021010239A1 (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7434742B2 (en) * 2019-07-22 2024-02-21 株式会社島津製作所 Nucleic acid detection method
JPWO2021193862A1 (en) * 2020-03-26 2021-09-30
WO2021200717A1 (en) * 2020-03-31 2021-10-07 東洋紡株式会社 Improved method of virus detection
WO2023176026A1 (en) * 2022-03-16 2023-09-21 株式会社島津製作所 Test method and test kit

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090123976A1 (en) * 2005-03-16 2009-05-14 Birnboim Chaim H Compositions and Method for Storage of Nucleic Acid From Bodily Fluids
JP2011078358A (en) * 2009-10-07 2011-04-21 Kyushu Univ Method for detecting norovirus

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0751080B2 (en) * 1991-02-21 1995-06-05 株式会社三和化学研究所 A simple method for detecting the human hepatitis C virus genome
JP2010046042A (en) * 2008-08-25 2010-03-04 Mukogawa Gakuin Method for detecting specific gene
WO2013002354A1 (en) * 2011-06-29 2013-01-03 株式会社ダナフォーム Biological sample pretreatment method, method for detecting rna, and pretreatment kit
WO2017088169A1 (en) * 2015-11-27 2017-06-01 Coyote Bioscience Co., Ltd. Methods and systems for nucleic acid amplification
JP2017209036A (en) * 2016-05-24 2017-11-30 東洋紡株式会社 Improved virus detection method

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090123976A1 (en) * 2005-03-16 2009-05-14 Birnboim Chaim H Compositions and Method for Storage of Nucleic Acid From Bodily Fluids
JP2011078358A (en) * 2009-10-07 2011-04-21 Kyushu Univ Method for detecting norovirus

Also Published As

Publication number Publication date
JPWO2021010239A1 (en) 2021-01-21
WO2021010239A1 (en) 2021-01-21
JP2023115278A (en) 2023-08-18

Similar Documents

Publication Publication Date Title
CN114080456A (en) RNA virus detection method
JP7167913B2 (en) Virus test method and virus test kit
Ko et al. Rapid detection of infectious adenoviruses by mRNA real-time RT-PCR
JP6642045B2 (en) Improved virus detection method
JP2021532770A (en) Methods and compositions for detecting microbial and viral particles
JP2017209036A (en) Improved virus detection method
JP2011520467A (en) Methods, kits and systems for collecting and processing samples for DNA and RNA detection
JP2018000138A (en) Improved virus detection method
JP2020156470A (en) Nucleic acid amplification method suppressed in non-specific amplification
JP2020018295A (en) Improved virus detection method
CN112029900A (en) Rapid nucleic acid detection method and detection system for novel coronavirus
JP2024026545A (en) Improved nucleic acid detection method
CN111254217A (en) Method for detecting norovirus
JP2023024808A (en) Methods for detecting rna viruses
US20210025012A1 (en) Method for detecting nucleic acid
JP2022191442A (en) Method for suppressing non-specific nucleic acid amplification
WO2021192320A1 (en) Examination method for novel coronavirus and examination reagent for same
JP2021019558A (en) Improved virus detection method
JP2021019559A (en) Improved virus detection method
WO2022085530A1 (en) Pretreatment method for preparing sample to be supplied for method for detecting foreign nucleic acid
WO2021020380A1 (en) Improved virus detection method
US20230287525A1 (en) Method for the concentration of microscopic substances derived from living organisms, environments, or foods
Sahu Tools and Techniques for Recovery, Detection, and Inactivation of Foodborne Viruses
WO2021193862A1 (en) Improved method of virus detection
WO2021200717A1 (en) Improved method of virus detection

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination