JP2020080807A - Methods for detecting norovirus - Google Patents

Abstract

To provide simple norovirus detection methods and kits for conducting the methods.SOLUTION: The invention relates to a method for detecting norovirus by reverse transcription-polymerase chain reaction (RT-PCR), and a kit for conducting the method. More specifically, the invention relates to a method for detecting norovirus and to a kit for conducting the method, where the method comprises mixing a test sample with a mixture of a sample processing fluid and an RT-PCR reaction solution, which mixture further contains dimethylsulfoxide.SELECTED DRAWING: None

Description

The present invention relates to a method for detecting norovirus by reverse transcription-polymerase chain reaction (RT-PCR), and a kit for carrying out the method. More specifically, Norovirus is detected by mixing the sample with the mixed solution of the sample treatment solution and the RT-PCR reaction solution, which further contains dimethyl sulfoxide (DMSO). A method and a kit for performing the method.

Norovirus is an RNA virus belonging to the human calicivirus family 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 under an electron microscope, and has also been called the generic name of Norwalk-like virus. Is. Viruses belonging to Norovirus are classified into two gene groups, Genogroup (GI) and Genogroup II (GII), and further classified into 14 and 17 or more genotypes, respectively.

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

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

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

WO2002/029119 WO2002/029120 Japanese Patent Laid-Open No. 2004-301684 JP, 2017-209036, A

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

An object of the present invention is to provide a simple method for detecting norovirus. Specifically, norovirus can be easily detected by mixing a sample with a mixed solution of a sample treatment solution and an RT-PCR reaction solution, which further contains dimethyl sulfoxide (DMSO). And a kit for carrying out the method.

The object of the present invention is achieved by the following inventions.
[1]
A method of detecting a norovirus in a sample by mixing the sample with a mixed solution of a sample treatment solution and a 1-step RT-PCR reaction solution, which further contains dimethyl sulfoxide (DMSO), and performing RT-PCR reaction. ..
[2]
The method according to [1], wherein the Norovirus genotype is Genogroup I (GI) or Genogroup II (GII).
[3]
The method according to [1] or [2], wherein the sample is derived from a sample selected from the group consisting of biological samples, biological samples, environmental samples, and environmental samples.
[4]
The method according to [1] or [2], wherein the specimen is derived from a sample selected from the group consisting of excrement samples, excrement-derived samples, vomitus and vomitus-derived samples.
[5]
The method according to [1] or [2], wherein the sample 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 sample is a centrifugal supernatant obtained by centrifuging the suspension according to [5].
[7]
The sample treatment solution is a Sample Treatment Reagent contained in a Norovirus detection reagent kit (probe method) (Shimadzu, product number 241-09325 series), and the 1-step RT-PCR reaction solution is NoV contained in the kit. The method according to any one of [1] to [6], which is a mixture of Reagents A, B, and C and is 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 liquid and the 1-step RT-PCR reaction liquid is 3:4 to 6 as a volume ratio.
[9]
The DMSO is contained in a mixed solution of the sample treatment solution and the 1-step RT-PCR reaction solution at a concentration of 1 to 5% (v/v), [1] to [8]. the method of.
[10]
The method according to any one of [1] to [9], wherein the mixing ratio of the sample and the mixed liquid further containing DMSO is 1:20 to 30 as a volume ratio.
[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 variants 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 variants 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 real-time measurement gives a result of determining whether the presence of Norovirus in the sample is positive or negative by measuring the amplification curve of the RT-PCR product using a fluorescent filter, [1] to The method according to any one of [13].
[15]
A sample treatment solution and a 1-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase are included, and 1 to 5% (v/v) is added to a mixed solution of the sample treatment solution and the 1-step RT-PCR reaction solution. A Norvirus Detection Kit configured to include DMSO.
[16]
The kit according to [15], wherein the norovirus genotype is determined to be genogroup I (GI) or genogroup II (GII).
[17]
Further, the kit according to [15] or [16], which further includes a procedure manual for the kit.

According to the present invention, a sample such as feces containing norovirus is directly added to a mixed solution of the sample treatment solution and the 1-step RT-PCR reaction solution, which further contains dimethyl sulfoxide (DMSO). , RNA release from Norovirus, RT-PCR reaction, and detection of RT-PCR products can be performed continuously in the same container, so that virus detection can be performed easily. Furthermore, the method of the present invention does not require the heat treatment that has been conventionally required in order to release RNA from the norovirus particles, and thus the simplicity is improved.

Amplification curve in real-time PCR performed by changing the concentration of DMSO added to the reaction system using the norovirus detection reagent kit (probe method) (Shimadzu) using the centrifugation supernatant of the suspension of feces containing norovirus as a sample. FIG. For 4 types of stool containing norovirus, the supernatant of the stool suspension was used as a sample, and 2% DMSO was added or not added to the reaction system using the norovirus detection reagent kit (probe method) (Shimadzu). It is a figure which shows the amplification curve in the performed real-time PCR.

The present invention provides a method for detecting Norovirus in a sample. In the method, a sample is mixed with a sample treatment solution and a 1-step RT-PCR reaction solution, which is further mixed with dimethyl sulfoxide (DMSO), and the RT-PCR reaction causes norovirus in the sample. Is a method of detecting.

In the present invention, the Norovirus to be detected in the sample is an RNA virus having RNA as a genome and not having an envelope which is a membrane composed of a lipid bilayer. Since the main component of the envelope is lipid, it is easily destroyed by an organic solvent such as alcohol or a surfactant, but RNA viruses such as Norovirus that do not have such an envelope are generally treated with an organic solvent or a surfactant. Shows resistance to.

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

In another embodiment of the present invention, the sample includes an excrement sample, an excrement-derived sample, a vomiting sample, a vomiting-derived sample, and the like. The excrement sample and the vomiting sample may be suspended in distilled water, physiological saline or a buffer solution at 5 to 10% (w/v) to prepare an emulsion, and this emulsion may be used as a sample. The buffer solution is not particularly limited, and examples thereof include a phosphate buffer solution, a Tris buffer solution, a borate buffer solution, and a Good buffer solution such as HEPES. The emulsion may be centrifuged at 10000 to 12000 rpm for 2 to 20 minutes, and the obtained centrifugation supernatant may be used as a sample.

Excretion-derived and vomit-derived samples include wipe samples. The wiping sample was obtained by wiping fingers, tableware, cutting boards, knives, cooking equipment, toilet equipment, housing equipment, etc. with a cotton swab, cut cotton, etc. to elute in a phosphate buffer solution, etc. for the purpose of confirming virus contamination. It is a thing. The obtained eluate is subjected to ultracentrifugation and the suspension or solution of the centrifugal sediment can be used as a sample (Keiko Munemura et al., Journal of Food Hygiene, Vol. 58, No. 4, p. 201-204, 2017). ..

The sample treatment solution and the 1-step RT-PCR reaction solution used in the present invention are commercially available norovirus detection reagent kits (probe method) (Shimadzu, product number 241-09325 series, 241-09325-91 or 241-09325. It is preferable to combine the reagents contained in -92). As the sample treatment liquid, the Sample Treatment Reagent included in this kit can be used. As the 1-step RT-PCR reaction solution, a mixture of NoV Reagents A, B and C contained in this kit can be used. NoV Reagent A contains magnesium ions, potassium ions and Tris. NoV Reagent B contains a reverse transcription reaction primer and a PCR primer. NoV Reagent C contains reverse transcriptase and DNA polymerase. In the one-step RT-PCR reaction, since the reverse transcriptase and the DNA polymerase are mixed in advance, the reverse transcription reaction (single-strand cDNA synthesis) and PCR can be performed in the same container. The mixing ratio of the sample treatment liquid and the 1-step RT-PCR reaction liquid is preferably 3:4 to 6 as a volume ratio, and more preferably 3:5 to 5.2.

The reverse transcriptase contained in the 1-step RT-PCR reaction solution is an enzyme that produces single-stranded complementary DNA (cDNA) using viral RNA as a template, and is not particularly limited as long as it catalyzes the reverse transcription reaction. Dependence of RNA Virus Derived from Avian Myeloblastosis Virus (AMV), Moloney Murine Leukemia Virus (M-MLV) and Human Immunodeficiency Virus (HIV) DNA polymerase as well as variants thereof can be used.

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

The 1-step RT-PCR reaction solution contains all components for performing reverse transcription reaction and PCR under appropriate conditions. The components include at least the reverse transcriptase, reverse transcription reaction primer, thermostable DNA polymerase, PCR primer, dNTP mix (deoxyribonucleotide 5'-triphosphate; mixture of dATP, dGTP, dCTP and dTTP) and a buffer solution. .. An RNA degrading enzyme inhibitor may be added to the reaction solution. As the reverse transcription reaction primer, a primer specific to the sequence of the target RNA, an oligo(dT) primer or a random primer can be used. As the PCR primers, a primer pair (forward and reverse) specific to the sequence of cDNA generated by the reverse transcription reaction is used. The PCR primer may be identical to the reverse transcription reaction primer specific for the sequence of the target RNA. Further, two or more kinds of PCR primers may be added to the one-step RT-PCR reaction solution depending on the number of DNA regions to be amplified, that is, target sequences. NoV Reagent A, B contained in a commercially available norovirus detection reagent kit (probe method) (Shimadzu, product number 241-09325 series, 241-09325-91 or 241-09325-92) as a composition containing the above components A mixture of C and C according to the kit instruction manual can be used as a 1-step RT-PCR reaction solution.

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

In the present invention, in order to release RNA from Norovirus, the step of performing heat treatment such as 90° C. which is conventionally required is not necessary. In the present invention, a sample can be directly added to a mixed solution of the sample treatment solution and the 1-step RT-PCR reaction solution, and norovirus can be detected by the RT-PCR reaction. This is possible 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 1-step RT-PCR reaction solution, or may be added to the mixed solution, as long as the final concentration is 1 to 5% (v/v). .. A more preferable final concentration of DMSO in the mixed solution is 2% (v/v).

In order to efficiently release RNA from Norovirus and to detect Norovirus by one-step RT-PCR reaction with good sensitivity, the mixing ratio of the sample and the mixed solution containing DMSO is 1:20 to a volume ratio. 30 is preferable and 1:25 is more preferable.

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).

In the present invention, in the RT-PCR reaction, the PCR product is monitored by real-time measurement. When performing the real-time measurement, RT-PCR and the step of detecting the RT-PCR product are performed in the same container.

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

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

In the real-time measurement of the PCR product, the amplification curve of the RT-PCR product is monitored using a fluorescent filter corresponding to the fluorescent dye used. When the fluorescence intensity increases according to the number of PCR cycles, the presence of the RNA virus to be analyzed in the sample is determined to be positive, while when the fluorescence intensity does not increase in PCR, it is determined to be negative. ..

In one embodiment of the present invention, a sample treatment solution and a 1-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase are included, and a mixture of the sample treatment solution and the 1-step RT-PCR reaction solution contains 1 A Norovirus detection kit configured to contain ˜5% (v/v) DMSO is provided.

Next, the present invention will be described in detail with reference to examples, but the scope of the present invention is not limited thereby.

[Effect of DMSO on detection of norovirus]
(1) Sample 100 mg of feces of a Norovirus-infected patient was collected and suspended in 1 mL of distilled water to prepare about 10% (w/v) fecal emulsion. The fecal emulsion was centrifuged at 10,000 rpm for 5 minutes with a microcentrifuge, and the obtained centrifugation supernatant was used as a sample.
(2) Preparation of reaction mixture The reaction mixture was prepared by mixing the reagents contained in the Norovirus detection reagent kit (probe method) (Shimadzu, product number 241-09325 series). The 1-step RT-PCR reaction solution was prepared by collecting 12.5 μL, 2.5 μL and 0.25 μL from NoV Reagent A, NoV Reagent B and NoV Reagent C of this kit, respectively, and mixing them. To this 1-step RT-PCR reaction solution, 9 μL was sampled from Sample Treatment Reagent of this kit as a sample treatment solution and added. DMSO was added to the mixed solution of the obtained sample treatment solution and the 1-step RT-PCR reaction solution so that the final concentration was 1, 2, 5 or 10% (v/v), and the reaction was carried out. A mixed solution was prepared.
(3) Mixing of sample and reaction solution Add 25 μL of the reaction mixture prepared in (2) to the PCR reaction tube, and add 1 μL of the sample obtained in (1) to the real-time PCR device (GVP-9600, The Shimadzu Corporation) was used to immediately monitor the RT-PCR reaction. The reaction mixture contained COG1F/COG1R and COG2F/COG2R as PCR primers and G1A, G1B and G2 as fluorescent labeled probes.
(4) RT-PCR conditions After reverse transcription reaction at 45°C for 5 minutes, initial denaturation at 95°C for 3 minutes was performed, and then PCR at 95°C for 1 second and -56°C for 10 seconds was performed for 45 cycles. Photometry in PCR was performed at a step of 56°C/10 seconds.
(5) Result and consideration The photometric result is shown in FIG. It was shown that the Ct value and the fluorescence intensity were influenced by the DMSO concentration. The Ct value is the number of cycles at which the amplification curve intersects the threshold value in real-time PCR. Compared with the case where DMSO was not added (0% DMSO), when 1, 2 and 5% (v/v) DMSO was added, the Ct value was small and a strong fluorescence intensity was obtained. This effect was most strongly observed with 2% DMSO. By adding 1 to 5% (v/v) DMSO to the reaction mixture, it was shown that the amount of initial DNA was larger than that in the case where DMSO was not added, and it was found that norovirus was detected.

[Effect of DMSO on different fecal samples]
(1) Specimens Four types of feces of Norovirus-infected patients were obtained by centrifugation in the same manner as in Example 1 and used as specimens.
(2) Preparation of Reaction Mixture The same procedure as in Example 1 was carried out except that the DMSO concentration was 2% (v/v) and 0%.
(3) Mixing of sample and reaction solution and RT-PCR
The same procedure as in Example 1 was performed.
(4) Result and consideration The photometric result is shown in FIG. It was shown that addition of 2% (v/v) DMSO to the reaction mixture resulted in a smaller Ct value and higher fluorescence intensity in all the measured feces than in the case of no addition of DMSO. Was done. Therefore, it can be seen that the method of the present invention enables simple detection of norovirus and identification of norovirus-infected patients.

Claims (17)

The sample is mixed with a sample treatment solution and a 1-step RT-PCR reaction solution, which further contains dimethylsulfoxide (DMSO), and the RT-PCR reaction detects norovirus in the sample. Method. The method of claim 1, wherein the Norovirus genotype is Genogroup I (GI) or Genogroup II (GII). The method according to claim 1 or 2, wherein the specimen is derived from a sample selected from the group consisting of biological samples, biological samples, environmental samples, and environmental samples. The method according to claim 1 or 2, wherein the specimen is derived from a sample selected from the group consisting of excrement samples, excrement-derived samples, vomitus and vomitus-derived samples. The method according to claim 1 or 2, wherein the sample is a suspension obtained by suspending the sample according to claim 4 in water, physiological saline, or a buffer solution. The method according to claim 1, wherein the sample is a centrifugation supernatant obtained by centrifuging the suspension according to claim 5. The sample treatment solution is a Sample Treatment Reagent contained in a Norovirus detection reagent kit (probe method) (Shimadzu, product number 241-09325 series), and the 1-step RT-PCR reaction solution is NoV contained in the kit. The method according to any one of claims 1 to 6, which is a mixture of Reagents A, B and C, which is a reaction solution containing a reverse transcriptase and a DNA polymerase. The method according to claim 1, wherein a mixing ratio of the sample treatment liquid and the 1-step RT-PCR reaction liquid is 3:4 to 6 as a volume ratio. 9. The DMSO is contained in a mixed solution of the sample treatment solution and the 1-step RT-PCR reaction solution at a concentration of 1 to 5% (v/v), according to claim 1. the method of. The method according to any one of claims 1 to 9, wherein a mixing ratio of the sample and the mixed liquid further containing DMSO is 1:20 to 30 as a volume ratio. The method according to claim 7, wherein the reverse transcriptase is selected from the group consisting of AMV reverse transcriptase, MMLV reverse transcriptase, HIV reverse transcriptase and variants thereof. The method according to 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 variants thereof. 13. The method according to any one of claims 1-12, wherein the RT-PCR reaction is monitored by real-time measurement. The real-time measurement gives a result of determining whether the presence of Norovirus in a sample is positive or negative by measuring an amplification curve of an RT-PCR product using a fluorescence filter. The method according to any one of 13 above. A sample treatment solution and a 1-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase are included, and 1 to 5% (v/v) is added to a mixed solution of the sample treatment solution and the 1-step RT-PCR reaction solution. A detection kit for Norvirus, configured to include DMSO. The kit according to claim 15, which determines that the Norovirus genotype is Genogroup I (GI) or Genogroup II (GII). The kit according to claim 15 or 16, further comprising a procedure manual for the kit.