JP7555179B2 - How to detect norovirus - Google Patents

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, the present invention relates to a method for detecting norovirus by mixing a sample with a mixture of a sample treatment solution and an RT-PCR reaction solution, the mixture further containing dimethyl sulfoxide (DMSO), and a kit for carrying out the method.

Norovirus is an RNA virus belonging to the human Caliciviridae family, with a genome of single-stranded RNA of approximately 7,000 bases. This virus is also called Small Round Structured Virus (SRSV) based on morphological classification observed under an electron microscope, and has also been referred to by the genus name Norwalk-like virus. Viruses belonging to the norovirus family are classified into two genogroups, Genogroup (GI) and Genogroup II (GII), which are further classified into 14 and 17 or more genotypes, respectively.

When norovirus infects humans, it causes acute gastroenteritis symptoms such as vomiting and diarrhea. Approximately half of the annual food poisoning cases in Japan are caused by norovirus, and of these, approximately 70% occur between November and February. Norovirus is known as the virus that causes winter-type gastroenteritis and food poisoning. Food poisoning caused by norovirus occurs mainly due to food contamination by food cooks. Norovirus is highly contagious and prone to mass outbreaks such as large-scale food poisoning. Humans are mainly infected through oral infection. Representative sources of infection include feces or vomit from infected individuals and items directly or indirectly contaminated by these, as well as foods such as oysters or other bivalve mollusks contaminated by norovirus. For this reason, it is important to identify norovirus-infected patients and items contaminated by the virus in order to prevent the spread of viral infection.

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

RNA virus particles have a basic structure in which a core consisting of an RNA genome and protein is enclosed in a protein shell called a capsid. Therefore, in order to detect viral RNA by gene amplification methods, it is necessary to extract RNA from the viral particles. To detect norovirus in feces as a specimen, for example, a fecal specimen is suspended in distilled water or physiological saline at a concentration of 5 to 10% (w/v), and RNA is extracted and purified from the centrifugal supernatant using a commercially available viral RNA extraction kit (e.g., QIAamp (registered trademark) Viral RNA Mini, QIAGEN) (Non-Patent Document 2). However, the detection process of performing RT-PCR after multiple steps of RNA extraction and purification is complicated. For this reason, a simple detection method has been proposed in which the fecal suspension is mixed with a specimen treatment solution, heat-treated for a short time to remove shell 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 a fecal suspension and a specimen treatment solution, the reaction vessel must be sealed with a lid to prevent the mixture from bumping or evaporating, and the lid must be removed after heat treatment to add the RT-PCR reaction solution, which is time-consuming. To improve this, a method has been proposed in which the specimen is mixed with a chaotropic agent such as a guanidine salt to detect viruses by RT-PCR without heat treatment (Patent Document 4).

WO2002/029119 WO2002/029120 JP2004-301684 Patent Publication No. 2017-209036

Kageyama T, et al. Broadly reactive and highly sensitive assay sensitive for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. J Clin Microbiol. 2003 Apr;41(4):1548-57. Ministry of Health, Labor and Welfare, Food Safety Bureau, Food Safety Inspection Division, Food Safety Inspection No. 1105001 (November 5, 2003) Attachment "Detection method of norovirus", last revised: Food Safety Inspection No. 0514004 (May 14, 2007) Ministry of Health, Labor and Welfare, Food Safety Inspection Division, Food Safety Department, Pharmaceuticals and Food Safety Bureau, Food Safety Inspection Notification No. 1105001 (November 5, 2003) with an attachment "Methods for detecting norovirus", last revised: Food Safety Inspection Notification No. 1022-1 (October 22, 2013) 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.

The object of the present invention is to provide a simple method for detecting norovirus. Specifically, the object is to provide a method for easily detecting norovirus by mixing a sample with a mixture of a sample treatment solution and an RT-PCR reaction solution, the mixture further containing 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 for detecting norovirus in a sample by RT-PCR reaction, comprising mixing the sample with a mixture of a sample treatment solution and a one-step RT-PCR reaction solution, the mixture further containing dimethyl sulfoxide (DMSO).
[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 specimen is derived from a sample selected from the group consisting of a biological sample, a biologically derived sample, an environmental sample, and an environmentally derived sample.
[4]
The method according to [1] or [2], wherein the specimen is derived from a sample selected from the group consisting of a fecal sample, a fecal-derived sample, vomit, and a vomit-derived sample.
[5]
The method according to [1] or [2], wherein the specimen is a suspension of 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 method according to any one of [1] to [6], wherein the sample treatment solution is a Sample Treatment Reagent included in a Norovirus Detection Reagent Kit (Probe Method) (Shimadzu Corporation, Product No. 241-09325 Series), and the one-step RT-PCR reaction solution is a mixture of NoV Reagents A, B, and C included in the kit, and is a reaction solution containing reverse transcriptase and DNA polymerase.
[8]
The method according to any one of [1] to [7], wherein a mixing ratio of the specimen treatment solution to the one-step RT-PCR reaction solution is 3:4 to 6 by volume.
[9]
The method according to any one of [1] to [8], wherein the DMSO is contained in a mixture of the sample treatment solution and the one-step RT-PCR reaction solution at a concentration of 1 to 5% (v/v).
[10]
The method according to any one of [1] to [9], wherein a mixing ratio of the sample to the mixed solution further containing DMSO is 1:20 to 1:30 in terms of 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 mutants 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 mutants 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 uses a fluorescent filter to measure an amplification curve of an RT-PCR product, thereby providing a result that determines whether the presence of norovirus in the sample is positive or negative.
[15]
A norvirus detection kit comprising a specimen treatment solution and a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase, and configured so that a mixture of the specimen treatment solution and the one-step RT-PCR reaction solution contains 1 to 5% (v/v) DMSO.
[16]
The kit according to [15], which determines whether the norovirus genotype is genogroup I (GI) or genogroup II (GII).
[17]
The kit according to [15] or [16], further comprising an instruction manual for operating the kit.

According to the present invention, by directly adding a specimen such as stool containing norovirus to a mixture of a specimen treatment solution and a one-step RT-PCR reaction solution that further contains dimethyl sulfoxide (DMSO), RNA release from norovirus, RT-PCR reaction, and detection of the RT-PCR product can be carried out consecutively in the same container, allowing for simple virus detection. Furthermore, the method of the present invention does not require the heat treatment that was previously required to release RNA from norovirus particles, making the method even simpler.

FIG. 1 shows amplification curves in real-time PCR performed using a centrifugal supernatant of a fecal suspension containing norovirus as a sample and a Norovirus detection reagent kit (probe method) (Shimadzu Corporation) at different concentrations of DMSO added to the reaction system. FIG. 1 shows amplification curves in real-time PCR for four types of feces containing norovirus, using the centrifugal supernatant of a fecal suspension as a sample and a Norovirus detection reagent kit (probe method) (Shimadzu Corporation) with or without the addition of 2% DMSO to the reaction system.

The present invention provides a method for detecting norovirus in a specimen. The method comprises mixing the specimen with a mixture of a specimen treatment solution and a one-step RT-PCR reaction solution, the mixture further containing dimethyl sulfoxide (DMSO), and detecting norovirus in the specimen by RT-PCR reaction.

In the present invention, the norovirus to be detected in a specimen is an RNA virus that has RNA as its genome and does not have an envelope, which is a membrane made of a lipid bilayer. Since the main component of the envelope is lipid, it is easily destroyed by organic solvents such as alcohol and surfactants, but such non-enveloped RNA viruses such as norovirus generally exhibit resistance to organic solvents and surfactants.

Specimens in the present invention include biological samples, biological samples, environmental samples, and environmental samples. Biological samples include animal and plant tissues, including the midgut gland of shellfish, and body fluids, such as blood, saliva, nasal secretions, and tissue secretions. Shellfish in particular are considered the most important food source for food poisoning caused by norovirus. Biological samples include the biological samples that have been treated, for example, by sonication. Environmental samples include all samples, including air, soil, dust, and water. Environmental samples include the environmental samples that have been treated, for example, by sonication.

In another embodiment of the present invention, the specimen may be a fecal sample, a fecal-derived sample, a vomit sample, or a vomit-derived sample. The fecal sample and the vomit sample may be suspended in distilled water, physiological saline, or a buffer solution at 5-10% (w/v) to form an emulsion, and this emulsion may be used as the specimen. The buffer solution may include, but is not limited to, phosphate buffer, Tris buffer, borate buffer, and Good's buffer such as HEPES. The emulsion may be centrifuged at, for example, 10,000-12,000 rpm for 2-20 minutes, and the resulting centrifugal supernatant may be used as the specimen.

Swab samples are included in samples derived from excrement and vomit. Swab samples are prepared by wiping hands, tableware, cutting boards, knives, cooking equipment, toilet equipment, and household equipment with a cotton swab or cotton wool, and then eluting the wipes in a phosphate buffer solution or similar for the purpose of checking for viral contamination. The eluate obtained is ultracentrifuged, and the centrifugal sediment is suspended or dissolved and can be used as a specimen (Munemura Yoshiko et al., Food Hygiene Journal, Vol. 58, No. 4, 2017, p. 201-204).

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

The reverse transcriptase contained in the one-step RT-PCR reaction solution is an enzyme that uses viral RNA as a template to generate single-stranded complementary DNA (cDNA), and is not particularly limited as long as it catalyzes the reverse transcription reaction. However, RNA-dependent DNA polymerases derived from RNA viruses such as Avian Myeloblastosis Virus (AMV), Moloney Murine Leukemia Virus (M-MLV), and Human Immunodeficiency Virus (HIV), as well as mutants thereof, can be used.

The DNA polymerase contained in the one-step RT-PCR reaction solution is a thermostable DNA polymerase derived from a thermophilic bacterium, and may be, but is not limited to, Taq, Tth, KOD, Pfu, or a mutant thereof. In order to avoid non-specific amplification by DNA polymerase, a hot-start DNA polymerase may be used. The hot-start DNA polymerase is, for example, a DNA polymerase bound to an anti-DNA polymerase antibody or a DNA polymerase whose enzyme active site has been chemically modified to be heat-sensitive, and is an enzyme that activates the DNA polymerase after the first denaturation step (90°C or higher) in PCR.

The one-step RT-PCR reaction solution contains all the components required for reverse transcription and PCR to be carried out under appropriate conditions. The components include at least the reverse transcriptase, the reverse transcription primer, the heat-resistant DNA polymerase, the PCR primer, the dNTP mix (a mixture of deoxyribonucleotide 5'-triphosphate; dATP, dGTP, dCTP, and dTTP), and a buffer solution. An RNase inhibitor can also 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 the cDNA generated by the reverse transcription reaction is used. The PCR primer may be the same as the reverse transcription primer specific to the sequence of the target RNA. In addition, two or more types of PCR primers may be added to the one-step RT-PCR reaction solution depending on the DNA region to be amplified, i.e., the number of target sequences. As a composition containing the above components, NoV Reagents A, B, and C included in a commercially available norovirus detection reagent kit (probe method) (Shimadzu Corporation, product numbers 241-09325 series, 241-09325-91 or 241-09325-92) can be mixed according to the kit's instructions and used as a one-step RT-PCR reaction solution.

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

In the present invention, a step of, for example, heat treatment at 90°C, which was previously required to liberate RNA from norovirus, is not required. In the present invention, a specimen can be directly added to a mixture of the specimen treatment solution and the one-step RT-PCR reaction solution, and norovirus can be detected by RT-PCR reaction. This is made possible by adding DMSO to the mixture, preferably at a final concentration of 1 to 5% (v/v). DMSO may be added to the specimen treatment solution or the one-step RT-PCR reaction solution, or to the mixture, as long as the final concentration is 1 to 5% (v/v). A more preferred final concentration of DMSO in the mixture is 2% (v/v).

In order to efficiently release RNA from norovirus and to perform sensitive norovirus detection by one-step RT-PCR, the volume ratio of the sample to the mixture containing DMSO is preferably 1:20 to 30, and more preferably 1:25.

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, the RT-PCR and the process of detecting the RT-PCR product are performed in the same vessel.

Real-time measurement of PCR products is also called real-time PCR. In real-time PCR, PCR amplified products are usually detected by fluorescence. Fluorescence detection methods include a method using an intercalating fluorescent dye and a method using a fluorescently labeled probe. SYBR (registered trademark) Green I is used as an intercalating fluorescent dye, but is not limited to this. Intercalating fluorescent dyes bind to double-stranded DNA synthesized by PCR and emit fluorescence when irradiated with excitation light. The amount of PCR amplified product produced can be measured by measuring the intensity of this fluorescence.

Examples of fluorescently labeled probes include, but are not limited to, TaqMan probes, Molecular Beacons, and cycling probes. The TaqMan probe is an oligonucleotide modified with a fluorescent dye at the 5' end and a quencher at the 3' end. The TaqMan probe hybridizes specifically 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 suppression of the generation of fluorescence by the quencher is released, causing the probe to emit fluorescence. The amount of the amplified product produced can be measured by measuring the intensity of this fluorescence. Examples of the fluorescent dye include, but are not limited to, FAM, ROX, and Cy5. Examples of the quencher include, but are not limited to, TAMRA (registered trademark) and MGB. To distinguish and detect two or more types of DNA target sequences, PCR is performed using two or more types of oligonucleotide probes (e.g., TaqMan probes) each bound to a different fluorescent dye.

In real-time measurement of PCR products, the amplification curve of the RT-PCR product is monitored using a fluorescence filter that corresponds to the fluorescent dye used. If the fluorescence intensity increases according to the number of PCR cycles, the presence of the RNA virus being analyzed in the sample is determined to be positive, whereas if the fluorescence intensity does not increase during PCR, the result is determined to be negative.

In one embodiment of the present invention, a norovirus detection kit is provided, which includes a specimen treatment solution and a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase, and is configured so that the mixture of the specimen treatment solution and the one-step RT-PCR reaction solution contains 1 to 5% (v/v) DMSO.

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

[Effect of DMSO on detection of norovirus]
(1) Samples 100 mg of feces from a patient infected with norovirus was collected and suspended in 1 mL of distilled water to prepare a fecal emulsion of about 10% (w/v). The fecal emulsion was centrifuged at 10,000 rpm for 5 minutes in a microcentrifuge, and the resulting centrifugal supernatant was used as a sample.
(2) Preparation of reaction mixture The reaction mixture was prepared by mixing the reagents included in the Norovirus Detection Reagent Kit (Probe Method) (Shimadzu Corporation, Product No. 241-09325 series). The one-step RT-PCR reaction solution was prepared by mixing 12.5 μL, 2.5 μL, and 0.25 μL of NoV Reagent A, NoV Reagent B, and NoV Reagent C from the kit. 9 μL of the Sample Treatment Reagent from the kit was added to the one-step RT-PCR reaction solution as a specimen treatment solution. DMSO was added to the mixture of the obtained specimen treatment solution and the one-step RT-PCR reaction solution to a final concentration of 1, 2, 5, or 10% (v/v), and mixed to prepare the reaction mixture.
(3) Mixing of the sample and the reaction solution 25 μL of the reaction mixture prepared in (2) was added to a PCR reaction tube, to which 1 μL of the sample obtained in (1) was added, and the RT-PCR reaction was immediately monitored using a real-time PCR device (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 reverse transcription at 45°C for 5 minutes, initial denaturation at 95°C for 3 minutes was performed, followed by 45 cycles of PCR at 95°C for 1 second and 56°C for 10 seconds. Photometry during PCR was performed at 56°C for 10 seconds.
(5) Results and Discussion The photometric results are shown in Figure 1. It was shown that the Ct value and fluorescence intensity were affected by the DMSO concentration. The Ct value is the cycle number at which the amplification curve crosses the threshold in real-time PCR. Compared to the case where no DMSO was added (0% DMSO), the Ct value was small and the fluorescence intensity was strong when 1, 2, and 5% (v/v) DMSO was added. This effect was most pronounced in the case of 2% DMSO. It was shown that the initial DNA amount was large by adding 1 to 5% (v/v) DMSO to the reaction mixture compared to the case where no DMSO was added, and norovirus was detected.

Effect of DMSO in different fecal samples
(1) Samples Four types of feces from patients infected with norovirus were centrifuged in the same manner as in Example 1 to obtain supernatants as samples.
(2) Preparation of reaction mixture The reaction mixture was prepared in the same manner as in Example 1, except that the DMSO concentration was 2% (v/v) and 0%.
(3) Mixing the sample with the reaction solution and RT-PCR
The procedure was the same as in Example 1.
(4) Results and Discussion The photometric results are shown in Figure 2. It was shown that in all feces samples measured, the addition of 2% (v/v) DMSO to the reaction mixture resulted in smaller Ct values and stronger fluorescence intensity than when DMSO was not added. Therefore, it is clear that the method of the present invention can easily detect norovirus and identify patients infected with norovirus.

Claims (16)

The method comprises mixing a specimen with a mixture of a specimen treatment solution and a one-step RT-PCR reaction solution, the mixture further containing dimethyl sulfoxide (DMSO), the DMSO being contained in the mixture of the specimen treatment solution and the one-step RT-PCR reaction solution at a concentration of 1 to 5% (v/v) , and detecting norovirus in the specimen by RT-PCR reaction. 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 a biological sample, a biologically derived sample, an environmental sample, and an environmentally derived sample. The method of claim 1 or 2, wherein the specimen is derived from a sample selected from the group consisting of a fecal sample, a fecal-derived sample, vomit, and a vomit-derived sample. The method according to claim 1 or 2, wherein the specimen is a suspension of the sample according to claim 4 in water, physiological saline or a buffer solution. The method according to claim 1 or 2, wherein the sample is a centrifugal supernatant obtained by centrifuging the suspension according to claim 5. The method according to any one of claims 1 to 6, wherein the one-step RT-PCR reaction solution is a reaction solution containing a reverse transcriptase and a DNA polymerase. 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 by volume. The method according to any one of claims 1 to 8, wherein the mixing ratio of the sample to the mixture further containing DMSO is 1:20 to 1:30 by volume. 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. 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 mutants thereof. The method according to any one of claims 1 to 11, wherein the RT-PCR reaction is monitored by real-time measurement. The method according to claim 12 , wherein the real-time measurement provides a result determining whether the presence of Norovirus in a specimen is positive or negative by measuring an amplification curve of an RT-PCR product using a fluorescent filter. A norovirus detection kit comprising a specimen treatment solution and a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase, wherein a mixture of the specimen treatment solution and the one-step RT-PCR reaction solution contains 1 to 5% (v/v) DMSO. The kit according to claim 14, which determines whether a norovirus genotype is genogroup I (GI) or genogroup II (GII). 16. The kit of claim 14 or 15, further comprising instructions for operating the kit.