CN111254217A - Method for detecting norovirus - Google Patents
Method for detecting norovirus Download PDFInfo
- Publication number
- CN111254217A CN111254217A CN201910995901.1A CN201910995901A CN111254217A CN 111254217 A CN111254217 A CN 111254217A CN 201910995901 A CN201910995901 A CN 201910995901A CN 111254217 A CN111254217 A CN 111254217A
- Authority
- CN
- China
- Prior art keywords
- sample
- norovirus
- kit
- solution
- pcr
- 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
Links
Images
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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2521/00—Reaction characterised by the enzymatic activity
- C12Q2521/10—Nucleotidyl transfering
- C12Q2521/107—RNA dependent DNA polymerase,(i.e. reverse transcriptase)
-
- 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
- C12Q2527/00—Reactions demanding special reaction conditions
- C12Q2527/125—Specific component of sample, medium or buffer
-
- 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
- C12Q2561/00—Nucleic acid detection characterised by assay method
- C12Q2561/113—Real time assay
-
- 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
- C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
- C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
Abstract
Provides a method for detecting norovirus. The present invention relates to a method for detecting norovirus using reverse transcription-polymerase chain reaction (RT-PCR), and a kit for performing the method. More specifically, the present invention relates to a method for detecting norovirus by mixing a sample with a mixed solution of a sample treatment solution and an RT-PCR reaction solution, the mixed solution further containing dimethyl sulfoxide, and a kit for carrying out the method.
Description
Technical Field
The present invention relates to a method for detecting norovirus using reverse transcription-polymerase chain reaction (RT-PCR), and a kit for performing the method. More specifically, a method for detecting norovirus by mixing a sample with a mixed solution of a sample treatment solution and an RT-PCR reaction solution, the mixed solution further containing dimethyl sulfoxide (DMSO), and a kit for carrying out the method.
Background
Norovirus is an RNA virus belonging to the family of human caliciviruses (human calicivirus) and has a single-stranded RNA of about 7000 bases in its genome. This Virus is also called Small Round Structured Virus (SRSV) according to the morphological classification observed with an electron microscope and is called a Virus under the generic name Norwalk-like Virus. Viruses belonging to norovirus are classified into 2 genomes, Genome (GI) and genome ii (gii), and further classified into 14 and 17 or more genotypes (genotype), respectively.
When a person infects norovirus, acute gastroenteritis symptoms such as vomiting and dysentery can be caused. About half of the annual food poisoning patients in japan are caused by norovirus, about 7 of which occur in 11 to 2 months, and norovirus is known as a causative virus of winter-type gastroenteritis and food poisoning. Food poisoning by norovirus occurs primarily from food contamination by the cook. Norovirus has strong infectivity and is easy to generate large-scale food poisoning and other group events. The route of infection in humans is primarily oral infection. Feces and vomit of infected persons, articles directly or indirectly contaminated with them, and foods such as oysters or other bivalve shellfish contaminated with norovirus are listed as typical sources of infection. Therefore, identification of norovirus infected patients, contaminants caused by the virus, is important to prevent expansion of viral infections.
As a virus test for detecting infection or contamination by a virus, an immunological measurement method for detecting a virus antigen or a virus gene amplification method is used (patent documents 1 to 3 and non-patent document 1). Examples of a means for detecting norovirus with high sensitivity include a method in which RNA of norovirus is amplified by RT-PCR and the amount of the amplified product is measured. For example, according to the notice of safety class monitoring by the department of food safety of the Ministry of health, labor and medicine (non-patent documents 2 and 3), norovirus detection by the RT-PCR method and quantitative norovirus detection by the real-time PCR method are widely performed.
RNA virions have a basic structure in which a core formed of an RNA genome and proteins is enclosed in 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 the supernatant is centrifuged to extract and purify RNA using a commercially available Viral RNA extraction kit (for example, QIAamp (registered trademark) Viral RNA Mini, QIAGEN corporation) (non-patent document 2). However, the detection process of RT-PCR after multi-stage RNA extraction/purification operation is complicated. Therefore, a simple detection method has been proposed in which a fecal suspension is mixed with a sample treatment solution, heat treatment is performed for a short period of time to remove the coat protein, the internal RNA is released, and the released 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 liquid for a specimen, the following work is required: in order to prevent bumping and evaporation of the mixture, the reaction vessel was sealed with a cap, and after the heat treatment, the cap was removed and the RT-PCR reaction solution was added. To improve this, a method of detecting a virus by RT-PCR without heat treatment by mixing a sample with a chaotropic agent such as a guanidine salt has been proposed (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 highly reactive for Norwalk-like viruses based on real-time quantitative conversion-PCR.J. Clin Microbiol.2003Apr; 41(4):1548-57.
Non-patent document 2: safety of food safety department of medicine and medicine from pachyson safety of food safety food safety at 3043512 from No. 1105001 (hei 15 years, 11 months and 5 days) was supplemented with "ノロウイルス estimate method について", finally made correct: food safety 304 0514004 (Flat 19 years 5 months and 14 days)
Non-patent document 3: safety of food safety department of medicine and medicine from pachyson safety of food safety food safety at 3043512 from No. 1105001 (hei 15 years, 11 months and 5 days) was supplemented with "ノロウイルス estimate method について", finally made correct: food elevator No. 1 (Flat 25 years 10 months 22 days)
Non-patent document 4: detection of noviruses in febrile 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 norovirus. Specifically, a method for easily detecting norovirus, in which a mixed solution of a sample treatment solution and an RT-PCR reaction solution, which further contains dimethyl sulfoxide (DMSO), is mixed with a sample to easily detect norovirus, and a kit for carrying out the method are provided.
Means for solving the problems
The object of the present invention is achieved by the following invention.
〔1〕
A method for detecting norovirus includes mixing a mixed solution of a treatment solution of a sample and a one-step RT-PCR reaction solution with the sample, and detecting norovirus in the sample through RT-PCR reaction, wherein the mixed solution further contains dimethyl sulfoxide (DMSO).
〔2〕
The method according to [ 1 ], wherein the norovirus genotype is Genome I (GI) or genome II (GII).
〔3〕
The method according to [ 1 ] or [ 2 ], 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.
〔4〕
The method according to [ 1 ] or [ 2 ], 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.
〔5〕
The method according to [ 1 ] or [ 2 ], wherein the specimen is a suspension obtained by suspending the sample according to [ 4 ] in water, physiological saline or a buffer solution.
〔6〕
The method according to [ 1 ] or [ 2 ], wherein the specimen is a centrifuged supernatant obtained by centrifuging the suspension according to [ 5 ].
〔7〕
The method according to any one of [ 1 ] to [ 6 ], wherein the sample treatment solution is a sample treatment Reagent (sample treatment Reagent) contained in a norovirus detection kit (Probe method) (Shimadzu corporation, product No. 241-09325 series), and the one-step RT-PCR reaction solution is a mixture of NoV reagents (NoVReagent) A, B and C contained in the kit and a reaction solution containing a reverse transcriptase and a DNA polymerase.
〔8〕
The method according to any one of [ 1 ] to [ 7 ], wherein a mixing ratio of the sample treatment solution to the one-step RT-PCR reaction solution is 3: 4 to 6.
〔9〕
The method according to any one of [ 1 ] to [ 8 ], wherein the DMSO is contained in a concentration of 1 to 5% (v/v) in a mixture of the sample treatment solution and the one-step RT-PCR reaction solution.
〔10〕
The method according to any one of [ 1 ] to [ 9 ], wherein a mixing ratio of the specimen to the mixed solution further containing DMSO is 1: 20 to 30.
〔11〕
The method according to [ 7 ], wherein the reverse transcriptase is selected from the group consisting of AMV reverse transcriptase, MMLV reverse transcriptase, HIV reverse transcriptase and a mutant thereof.
〔12〕
The method according to [ 7 ], 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.
〔13〕
The method according to any one of [ 1 ] to [ 12 ], wherein the RT-PCR reaction is monitored by real-time measurement.
〔14〕
The method according to any one of [ 1 ] to [ 13 ], wherein the real-time measurement is performed by measuring an amplification curve of an RT-PCR product using a fluorescence filter, and the presence of norovirus in the sample is determined to be positive or negative.
〔15〕
A detection kit for norovirus, which is composed of: which comprises the following steps: a sample treatment solution and a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase, wherein the mixed solution of the sample treatment solution and the one-step RT-PCR reaction solution contains 1-5% (v/v) of DMSO.
〔16〕
The kit according to [ 15 ], wherein the norovirus genotype is determined to be Genome I (GI) or genome II (GII).
〔17〕
The kit according to [ 15 ] or [ 16 ], which further comprises instructions for the procedures of the kit.
ADVANTAGEOUS EFFECTS OF INVENTION
According to the present invention, by directly adding a sample for stool containing norovirus to a mixed solution of a sample treatment solution and a one-step RT-PCR reaction solution, RNA release from norovirus, RT-PCR reaction, and detection of RT-PCR products can be continuously performed in the same container, and thus virus detection can be easily performed, the mixed solution further containing dimethyl sulfoxide (DMSO). Furthermore, in the method of the present invention, a heat treatment which is always necessary is not necessary for releasing RNA from norovirus particles, and the simplicity is further improved.
Drawings
FIG. 1 is a diagram showing an amplification curve in real-time PCR performed by using a norovirus detection kit (Probe method) (Shimadzu corporation) with the concentration of DMSO added to the reaction system being changed, using a centrifuged supernatant of a feces suspension containing norovirus as a sample.
FIG. 2 is a graph showing amplification curves in real-time PCR performed with a norovirus detection kit (Probe method) (Shimadzu corporation) using a centrifugal supernatant of a feces suspension as a specimen, with or without 2% DMSO being added to the reaction system for 4 kinds of feces containing norovirus.
Detailed Description
The present invention provides a method for detecting norovirus in a sample. The method for detecting norovirus comprises the following steps: and mixing a mixed solution of the treatment solution of the sample to be detected and the one-step RT-PCR reaction solution with the sample to be detected, and detecting the norovirus in the sample to be detected through RT-PCR reaction, wherein the mixed solution also contains dimethyl sulfoxide (DMSO).
In the present invention, norovirus to be detected in a sample is an RNA virus having RNA as a genome and does not have an envelope, which is a membrane formed of a lipid bilayer. 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 is most important as a food product causing food poisoning by norovirus. Examples of the biological sample include a sample obtained by subjecting the biological sample to a treatment such as an ultrasonic treatment. Examples of the environmental sample include all samples containing air, soil, dust, water, and the like. Examples of the environmental source sample include samples 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, a vomit-derived sample, and the like. The fecal sample and vomit sample may be suspended in distilled water, physiological saline or buffer solution at 5-10% (w/v) to prepare an emulsion, and the emulsion may be used as a sample. The buffer solution is not particularly limited, and examples thereof include phosphate buffer, Tris buffer, borate buffer, and zwitterionic (Good) buffer such as HEPES. The emulsion may be centrifuged at 10000 to 12000rpm for 2 to 20 minutes, for example, and the obtained centrifuged supernatant may be used as a sample.
The fecal and vomitus samples include swab samples. The wipe sample is a sample obtained by dissolving a substance wiped from fingers, tableware, chopping board, kitchen knife, cooking equipment, toilet equipment, housing equipment, etc. with a cotton swab, sliced cotton, etc. with a phosphate buffer solution, etc., for the purpose of confirming virus contamination. A substance obtained by subjecting the obtained eluate to ultracentrifugation and suspending or dissolving a centrifugal precipitate can be used as a sample (chazuki et al, food , No. 4 p.201-204, vol 58, 2017.
The treatment solution to be tested and the one-step RT-PCR reaction solution used in the present invention are preferably combined with reagents contained in a commercially available norovirus detection kit (probe method) (Shimadzu corporation, product No. 241-09325 series, 241-09325-91 or 241-09325-92). The Sample Treatment solution may be a Sample Treatment Reagent (Sample Treatment Reagent) contained in the kit. As the one-step RT-PCR reaction solution, a mixture of NoV reagent A, B and C contained in the kit can be used. NoV reagent A contains magnesium ions, potassium ions and Tris. NoV reagent B contains reverse transcription primers and PCR primers. NoV reagent C contains reverse transcriptase and DNA polymerase. In the one-step RT-PCR reaction, reverse transcriptase and DNA polymerase are mixed in advance, whereby reverse transcription reaction (single-stranded cDNA synthesis) and PCR can be performed in the same vessel. The mixing ratio of the sample treatment solution to the one-step RT-PCR reaction solution is preferably 3: 4-6, more preferably 3: 5 to 5.2.
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 catalyzes a reverse transcription reaction, and RNA-dependent DNA polymerases derived from RNA viruses such as Avian Myeloblastosis viruses (Avian Myeloblastosis Virus, AMV), Moloney murine Leukemia viruses (Moloney Murine Leukemia Virus, M-MLV) and Human Immunodeficiency viruses (Human Immunodeficiency Virus, HIV) and mutants thereof can be used.
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 mutants thereof 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 obtained by heat-sensitively chemically modifying an enzyme active site, and is an enzyme in which the DNA polymerase is activated after the initial denaturation step (90 ℃ or higher) in PCR.
The one-step RT-PCR reaction solution contains all the components for performing the reverse transcription reaction and PCR under appropriate conditions. The component includes at least the reverse transcriptase, a reverse transcription primer, the thermostable DNA polymerase, a PCR primer, a dNTP mixture (a mixture containing deoxynucleoside 5' -triphosphate, dATP, dGTP, dCTP and dTTP), and a buffer solution. An RNase inhibitor may be added to the reaction solution. As the reverse transcription 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 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 can be added to the one-step RT-PCR reaction solution depending on the number of amplified DNA regions, i.e., target sequences. As the composition containing the above-mentioned components, a mixture obtained by mixing the NoV reagent A, B and C contained in a commercially available norovirus detection kit (probe method) (Shimadzu corporation, product Nos. 241-09325 series, 241-09325-91, or 241-09325-92) according to the kit processing instructions can be used as the one-step RT-PCR reaction solution.
In the case of detecting norovirus RNA, the Genome I (GI) and the genome II (GII) in norovirus genotypes can be detected by using, for example, PCR primers described in patent documents 1 and 2, non-patent document 3, and Japanese patent laid-open publication No. 2018-78806, but not limited thereto. The norovirus detection kit (probe method) includes a PCR primer described in non-patent document 3.
In the present invention, a step of performing a heat treatment at 90 ℃ which is always necessary for releasing RNA from norovirus, for example, is not necessary. In the present invention, a sample is directly added to a mixed solution of the sample treatment solution and the one-step RT-PCR reaction solution, and norovirus can be detected by RT-PCR. This can be achieved by adding DMSO to the mixture, preferably at a final concentration of 1-5% (v/v). DMSO may be added to the sample treatment solution or the one-step RT-PCR reaction solution, or may be added to the mixture solution, as long as the final concentration of DMSO is 1 to 5% (v/v). The DMSO concentration in the mixture is more preferably 2% (v/v).
In order to efficiently release RNA from norovirus or to detect norovirus in a one-step RT-PCR reaction with good sensitivity, the mixing ratio of the sample to be tested to the mixed solution containing DMSO is preferably 1: 20-30, more preferably 1: 25.
the reaction temperature conditions for the reverse transcription reaction in RT-PCR and the PCR conditions (temperature, time and cycle number) can be easily set by those skilled in the art.
In the present invention, in the RT-PCR reaction, the PCR product is monitored by real-time measurement. In the real-time measurement, RT-PCR and the procedure for detecting the RT-PCR product are carried out in the same vessel.
Real-time determination of PCR products is also referred to as real-time PCR. In real-time PCR, PCR amplification products are generally 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 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 cycling 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 can specifically hybridize to the template DNA in the annealing step of PCR, but since a quencher is present on the probe, the occurrence of fluorescence is suppressed even when excitation light is irradiated thereto. In the subsequent extension reaction step, when the TaqMan probe hybridized with the template DNA is decomposed by the 5 '→ 3' exonuclease activity of the Taq DNA polymerase, the fluorescent dye is released from the probe, and the inhibition of the fluorescence generation by the quencher is released, so that fluorescence is emitted. 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 by discrimination, PCR is carried out using 2 or more oligonucleotide probes (e.g., TaqMan probes) to which different fluorescent dyes are bound, respectively.
In the real-time measurement of PCR products, the amplification curve of RT-PCR products was monitored using a fluorescent filter corresponding to the fluorescent dye used. When the fluorescence intensity increases according to the number of PCR cycles, the specimen is judged to be positive for the presence of the RNA virus to be analyzed, while when the fluorescence intensity does not increase during PCR, the specimen is judged to be negative.
In one embodiment of the present invention, there is provided a kit for detecting norovirus, comprising: which comprises the following steps: a sample treatment solution and a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase, wherein the mixed solution of the sample treatment solution and the one-step RT-PCR reaction solution contains 1-5% (v/v) of DMSO.
Examples
The present invention will be described in detail with reference to examples, but the scope of the present invention is not limited thereto.
Example 1
[ Effect of DMSO in norovirus assay ]
(1) Sample to be tested
100mg of feces of a patient infected with norovirus was 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 by a microcentrifuge, and the obtained centrifugal supernatant was used as a specimen.
(2) Preparation of reaction mixture
The reaction mixture was prepared by mixing the reagents contained in the norovirus detection kit (probe method) (Shimadzu corporation, product No. 241-09325 series). For the one-step RT-PCR reaction solution, 12.5. mu.L, 2.5. mu.L and 0.25. mu.L of the NoV reagent A, NoV reagent B and NoV reagent C of the kit were collected, respectively, and mixed to prepare a reaction solution. 9. mu.L of the sample treatment reagent of this kit was collected as a sample treatment solution and added to the one-step RT-PCR reaction solution. DMSO is added to and mixed with the obtained mixture of the sample treatment solution and the one-step RT-PCR reaction solution so that the final concentration of the mixture is 1%, 2%, 5% or 10% (v/v), thereby preparing a reaction mixture.
(3) Mixing of sample to be tested and reaction solution
mu.L of the reaction mixture prepared in (2) was added to a PCR reaction tube, 1. mu.L of the specimen obtained in (1) was added thereto, and the RT-PCR reaction was directly monitored using a real-time PCR apparatus (GVP-9600, Shimadzu corporation). The reaction mixture contained COG1F/COG1R and COG2F/COG2R as PCR primers, and G1A, G1B and G2 as fluorescently labeled probes.
(4) RT-PCR conditions
After a reverse transcription reaction at 45 ℃/5 min, initial denaturation at 95 ℃/3 min was performed, followed by PCR at 95 ℃/1 sec-56 ℃/10 sec for 45 cycles. Photometry in PCR was performed in steps of 56 ℃/10 sec.
(5) Results and investigation
The photometric results are shown in fig. 1. Indicating that Ct values and fluorescence intensities are affected by DMSO concentrations. The Ct value refers to the number of cycles that the amplification curve crosses the Threshold (Threshold) in real-time PCR. In the case where 1%, 2% and 5% (v/v) DMSO was added, the Ct value was small and a strong fluorescence intensity was obtained, compared with the case where DMSO was not added (0% DMSO). It was confirmed that the effect was strongest in the case of 2% DMSO. By adding 1-5% (v/v) DMSO to the reaction mixture, the amount of original DNA was larger than that in the case where DMSO was not added, and it was found that norovirus was detected.
Example 2
[ Effect of DMSO in different fecal specimens ]
(1) Sample to be tested
4 types of feces of patients infected with norovirus were treated in the same manner as in example 1, and centrifugation supernatants were obtained as specimens.
(2) Preparation of reaction mixture
The procedure of example 1 was repeated except that the DMSO concentrations were changed to 2% (v/v) and 0%.
(3) Mixing of sample and reaction solution and RT-PCR
The procedure was carried out in the same manner as in example 1.
(4) Results and investigation
The photometric results are shown in fig. 2. In all of the stools measured, 2% (v/v) DMSO was added to the reaction mixture, thereby showing that the Ct value was small and a strong fluorescence intensity was obtained as compared with the case where DMSO was not added. Therefore, it is found that norovirus can be easily detected by the method of the present invention, and a patient infected with norovirus can be identified.
Claims (17)
1. A method for detecting norovirus includes mixing a mixed solution of a treatment solution of a sample and a one-step RT-PCR reaction solution with the sample, and detecting norovirus in the sample through RT-PCR reaction, wherein the mixed solution further contains dimethyl sulfoxide (DMSO).
2. The method of claim 1, wherein the norovirus genotype is genome i (gi) or genome ii (gii).
3. The method according to claim 1 or 2, wherein the sample is derived from a sample selected from the group consisting of a biological sample, a sample of biological origin, an environmental sample and a sample of environmental origin.
4. The method according to claim 1 or 2, 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.
5. The method according to claim 1 or 2, wherein the specimen is a suspension obtained by suspending the sample according to claim 4 in water, physiological saline or a buffer solution.
6. The method according to claim 1 or 2, wherein the specimen is a centrifuged supernatant obtained by centrifuging the suspension according to claim 5.
7. The method according to any one of claims 1 to 6, wherein the Sample Treatment solution is a Sample Treatment Reagent (Sample Treatment Reagent) contained in a norovirus detection kit using a probe method, the one-step RT-PCR reaction solution is a mixture of NoV reagents (NoV Reagent) A, B and C contained in the kit, and is a reaction solution containing reverse transcriptase and DNA polymerase, and the kit is a kit of product No. 241-09325 series manufactured by Shimadzu corporation.
8. The method according to any one of claims 1 to 7, wherein the mixing ratio of the sample treatment solution to the one-step RT-PCR reaction solution is 3: 4 to 6.
9. The method according to any one of claims 1 to 8, wherein the DMSO is contained in a concentration of 1 to 5% (v/v) in a mixture of the sample treatment solution and the one-step RT-PCR reaction solution.
10. The method according to any one of claims 1 to 9, wherein a mixing ratio of the specimen to the mixed solution further containing DMSO is 1: 20 to 30.
11. The method of claim 7, wherein the reverse transcriptase is selected from the group consisting of AMV reverse transcriptase, MMLV reverse transcriptase, HIV reverse transcriptase, and mutants thereof.
12. The method of claim 7, 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.
13. The method of any one of claims 1 to 12, wherein the RT-PCR reaction is monitored by real-time assay.
14. The method according to any one of claims 13, wherein the real-time assay gives a result of determining the presence of norovirus in the sample as positive or negative by measuring an amplification curve of the RT-PCR product using a fluorescent filter.
15. A detection kit for norovirus, which is composed of:
which comprises the following steps: the kit comprises a to-be-detected sample treatment solution and a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase, wherein a mixed solution of the to-be-detected sample treatment solution and the one-step RT-PCR reaction solution contains 1-5% (v/v) of DMSO.
16. The kit of claim 15, which determines the norovirus genotype as either genome i (gi) or as genome ii (gii).
17. The kit of claim 15 or 16, further comprising instructions for the kit's procedures.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018-224895 | 2018-11-30 | ||
| JP2018224895A JP2020080807A (en) | 2018-11-30 | 2018-11-30 | Methods for detecting norovirus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111254217A true CN111254217A (en) | 2020-06-09 |
Family
ID=70904447
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910995901.1A Pending CN111254217A (en) | 2018-11-30 | 2019-10-18 | Method for detecting norovirus |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JP2020080807A (en) |
| KR (1) | KR20200066164A (en) |
| CN (1) | CN111254217A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPWO2021193862A1 (en) * | 2020-03-26 | 2021-09-30 | ||
| EP4060599A4 (en) | 2020-06-02 | 2023-06-14 | LG Energy Solution, Ltd. | Battery service providing system and method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726618A (en) * | 2015-04-15 | 2015-06-24 | 北京凯思百奥科技发展有限公司 | Norovirus detection kit and application thereof |
| JP2017131164A (en) * | 2016-01-28 | 2017-08-03 | 東洋紡株式会社 | Improved virus detection method |
| JP2018108055A (en) * | 2017-01-05 | 2018-07-12 | 東洋紡株式会社 | Oligonucleotide probe for detecting Norovirus G2 type |
| WO2018198682A1 (en) * | 2017-04-26 | 2018-11-01 | 東洋紡株式会社 | Virus test method and virus test kit |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002029120A1 (en) | 2000-09-29 | 2002-04-11 | Bml, Inc. | Method of detecting norwalk-like virus (gii) |
| EP1329522B2 (en) | 2000-09-29 | 2016-09-28 | BML, Inc. | Method of detecting norwalk-like virus (gi) |
| JP4300321B2 (en) * | 2002-10-25 | 2009-07-22 | 東洋紡績株式会社 | Method for promoting DNA synthesis reaction and composition therefor |
| JP3887340B2 (en) | 2003-03-31 | 2007-02-28 | デンカ生研株式会社 | Norovirus or Sapovirus specimen dilution and virus detection reagent |
| JP4719455B2 (en) * | 2004-12-07 | 2011-07-06 | シスメックス株式会社 | Biological sample treatment solution for direct nucleic acid amplification method and direct nucleic acid amplification method |
| EP2839275A1 (en) * | 2012-04-19 | 2015-02-25 | Wako Pure Chemical Industries Limited | Methods for real-time sampling of reaction products |
| JP2017209036A (en) | 2016-05-24 | 2017-11-30 | 東洋紡株式会社 | Improved virus detection method |
-
2018
- 2018-11-30 JP JP2018224895A patent/JP2020080807A/en active Pending
-
2019
- 2019-10-18 CN CN201910995901.1A patent/CN111254217A/en active Pending
- 2019-10-24 KR KR1020190132779A patent/KR20200066164A/en not_active Application Discontinuation
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726618A (en) * | 2015-04-15 | 2015-06-24 | 北京凯思百奥科技发展有限公司 | Norovirus detection kit and application thereof |
| JP2017131164A (en) * | 2016-01-28 | 2017-08-03 | 東洋紡株式会社 | Improved virus detection method |
| JP2018108055A (en) * | 2017-01-05 | 2018-07-12 | 東洋紡株式会社 | Oligonucleotide probe for detecting Norovirus G2 type |
| WO2018198682A1 (en) * | 2017-04-26 | 2018-11-01 | 東洋紡株式会社 | Virus test method and virus test kit |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20200066164A (en) | 2020-06-09 |
| JP2020080807A (en) | 2020-06-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7167913B2 (en) | Virus test method and virus test kit | |
| JP6642045B2 (en) | Improved virus detection method | |
| Stals et al. | Molecular detection and genotyping of noroviruses | |
| CN114080456A (en) | RNA virus detection method | |
| CN108330210B (en) | Zika virus, dengue virus and chikungunya virus nucleic acid detection kit and application thereof | |
| JP2021532770A (en) | Methods and compositions for detecting microbial and viral particles | |
| JP2017209036A (en) | Improved virus detection method | |
| JP7015288B2 (en) | Compositions and Methods for Detecting Human Parvovirus Nucleic Acids and Hepatitis A Virus Nucleic Acids in Singlex or Multiplex Assays | |
| JP2018000138A (en) | Improved virus detection method | |
| JP2020018295A (en) | Improved virus detection method | |
| CN112029900A (en) | Rapid nucleic acid detection method and detection system for novel coronavirus | |
| CN111254217A (en) | Method for detecting norovirus | |
| CN109182600B (en) | Fluorescent quantitative PCR kit for synchronously detecting hepatitis B virus, hepatitis C virus and human immunodeficiency virus type 1 | |
| JP2023024808A (en) | Methods for detecting rna viruses | |
| Dhar et al. | Detection and quantification of infectious hematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss) by SYBR Green real-time reverse transcriptase-polymerase chain reaction | |
| JP2011078358A (en) | Method for detecting norovirus | |
| US20210025012A1 (en) | Method for detecting nucleic acid | |
| Liu et al. | Development of reverse transcription loop-mediated isothermal amplification for rapid detection of Batai virus in cattle and mosquitoes | |
| JP7188645B2 (en) | Method for suppressing non-specific nucleic acid amplification | |
| Sung et al. | Reverse transcription loop-mediated isothermal amplification for rapid and sensitive detection of nervous necrosis virus in groupers | |
| WO2021193853A1 (en) | Test method and test reagent for novel coronavirus | |
| JP6592697B2 (en) | Serotype 2 dengue virus detection kit, serotype 3 dengue virus detection kit, and dengue virus detection kit | |
| CN107385116B (en) | Method for detecting type 1 human immunodeficiency virus and special reagent set thereof | |
| JP2021019559A (en) | Improved virus detection method | |
| JP2021019558A (en) | Improved virus detection method |
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 |