Oligonucleotides for detecting norovirus and uses thereof

Abstract

To provide useful methods for detecting norovirus, or the like.SOLUTION: Disclosed is a probe for detecting norovirus GI having the following characteristics (A) and (B), or a probe for detecting norovirus GII having the following characteristics (C) and (D): (A) the probe has a base sequence A1 of at least 15 consecutive bases in the base sequences at positions 10th to 85th of a specific sequence or its complementary base sequence, or a base sequence A2 in which 1 to 7 bases are substituted, deleted, inserted, or added in the base sequence A1; (B) only either 5' end or 3' end is labeled; (C) the probe has a base sequence C1 of at least 15 consecutive bases in the base sequences at positions 30th to 95th of the specific sequence or its complementary base sequence, or a base sequence C2 in which 1 to 7 bases are substituted, deleted, inserted, or added in the base sequence C1; and (D) only either the 5' end or the 3' end is labeled.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to oligonucleotides and the like for detecting norovirus (Norovirus: NV) that may be contained in a sample. Furthermore, the present invention relates to a method for detecting norovirus that may be contained in a sample using the oligonucleotides, and reagents and kits and the like for use in the method. [Background technology]

[0002] Norovirus is the main cause of viral gastroenteritis, and is prevalent in the winter, peaking between December and January each year. It is the most common food poisoning disease in Japan, accounting for more than 30% of all food poisoning cases annually. Norovirus multiplies in the human intestinal tract and is excreted in feces and vomit, so it is often found in toilets. Norovirus can be contracted by eating raw bivalve shellfish containing the virus or by eating food contaminated by a cook infected with the virus.

[0003] Furthermore, noroviruses are classified into five groups, GI to GV, based on their genotype, but it is mainly GI and GII that infect humans. Regarding norovirus testing, the Norovirus Pathogen Detection Manual published by the National Institute of Infectious Diseases also discloses a real-time PCR method using double-labeled nucleic acid probes (also called Taqman probes, hydrolysis probes, etc.) for the purpose of detecting GI and GII (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] National Institute of Infectious Diseases, Pathogen Detection Manual Norovirus, 1st Edition, released in June 2019 Summary of the Invention [Problem to be solved by the invention]

[0005] The real-time PCR method described in Non-Patent Document 1 is capable of quantitative detection, but since photometry is required for each PCR cycle, detection takes approximately one hour even in the shortest time.

[0006] Melting curve analysis is a known method for detecting nucleic acid amplification products. Melting curve analysis allows nucleic acid amplification and detection to be performed in separate steps, allowing for relatively simple measurements in as little as 30 minutes. Furthermore, melting curve analysis using fluorescently labeled nucleic acid probes has the advantage of being easily adaptable to genetic testing using automated analyzers. However, no nucleic acid probes capable of detecting norovirus GI and / or GII in a single reaction with high sensitivity using melting curve analysis have been identified.

[0007] One object of the present invention is to provide a useful method for detecting the GI and / or GII of norovirus that may be contained in a sample. [Means for solving the problem]

[0008] As a result of intensive research to achieve the above object, the present inventors have discovered a useful method for detecting the GI and / or GII of norovirus (particularly for simultaneously detecting GI and GII in one reaction), a particularly simple and highly sensitive method, by using a specific nucleic acid probe. Based on this finding, further investigation led to the completion of the present invention.

[0009] The present invention includes the following aspects. [Item 1] A probe for detecting GI of norovirus, having the following characteristics (A) and (B): (A) It has a base sequence A1 of at least 15 consecutive bases in the base sequence of positions 10 to 85 of SEQ ID NO: 1 or its complementary base sequence, or a base sequence A2 in which 1 to 7 bases have been substituted, deleted, inserted or added in base sequence A1. (B) Only either the 5' or 3' end is labeled. [Item 2] The probe according to Item 1, wherein the length of the base sequence of (A) is 15 to 30 bases. [Item 3] The probe according to Item 1 or 2, wherein the base sequence of (A) comprises a base sequence A3 shown in any one of SEQ ID NOs: 2 to 10 or a complementary base sequence A4 thereof, or a base sequence A5 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence A3 or A4. [Item 4] The probe according to any one of Items 1 to 3, wherein the label (B) is a fluorescent dye label. [Item 5] The probe according to any one of Items 1 to 4, wherein the label (B) is a label with a fluorescence quenching dye that is quenched when bound to a nucleic acid containing a base sequence that is 70% or more identical to a base sequence complementary to the base sequence of the probe. [Item 6] The probe according to any one of Items 1 to 5, wherein the label (B) is a label with a fluorescence quenching dye that is quenched by interaction with guanine. [Item 7] The probe according to any one of Items 1 to 6, wherein the label (B) is a label with at least one fluorescence quenching dye selected from the group consisting of fluorescein and its derivatives, rhodamine and its derivatives, and BODIPY and its derivatives. [Item 8] The probe according to any one of Items 1 to 7, wherein the label (B) is a label with at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue). [Item 9] The probe according to any one of Items 1 to 8, wherein in (B) above, the labeled terminal base is cytosine. [Item 10] A probe for detecting GII of norovirus, having the following characteristics (C) and (D): (C) It has a base sequence C1 of at least 15 consecutive bases in the base sequence 30 to 95 of SEQ ID NO: 11 or its complementary base sequence, or a base sequence C2 in which 1 to 7 bases have been substituted, deleted, inserted or added in the base sequence C1. (D) Only either the 5' or 3' end is labeled. [Item 11] The probe according to Item 10, wherein the length of the base sequence of (C) is 15 to 25 bases. [Item 12] The probe according to Item 10 or 11, wherein the base sequence of (C) comprises a base sequence C3 shown in any one of SEQ ID NOs: 12 to 15 or a complementary base sequence C4 thereof, or a base sequence C5 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence C3 or C4. [Item 13] The probe according to any one of Items 10 to 12, wherein the label (D) is a fluorescent dye label. [Item 14] The probe according to any one of Items 10 to 13, wherein the label (D) is a label with a fluorescence quenching dye that is quenched when bound to a nucleic acid containing a base sequence that is 70% or more identical to a base sequence complementary to the base sequence of the probe. [Item 15] The probe according to any one of Items 10 to 14, wherein the label (D) is a label with a fluorescence quenching dye that is quenched by interaction with guanine. [Item 16] The probe according to any one of Items 10 to 15, wherein the label (D) is a label with at least one fluorescence quenching dye selected from the group consisting of fluorescein and derivatives thereof, rhodamine and derivatives thereof, and BODIPY and derivatives thereof. [Item 17] The probe according to any one of Items 10 to 16, wherein the label (D) is a label with at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue). [Item 18] The probe according to any one of Items 10 to 17, wherein in (D), the labeled terminal base is cytosine. [Item 19] A method for detecting GI and / or GII of norovirus that may be contained in a sample in a single reaction using the probe according to any one of Items 1 to 9 and the probe according to any one of Items 10 to 18. [Item 20] The following steps (1), (2), and (3): (1) providing a sample that may contain GI and / or GII of norovirus; (2) performing a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1); and (3) detecting one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) using one or more probes according to any one of items 1 to 9 and one or more probes according to any one of items 10 to 18; Item 20. The method according to Item 19, comprising: [Item 21] The method according to Item 20, wherein step (2) is carried out by PCR reaction, and the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to family B. [Item 22] The method according to Item 21, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from KOD or a mutant thereof. [Item 23] The method of any of Items 20 to 22, wherein the norovirus GI detection primer set used in the nucleic acid amplification reaction of step (2) comprises a first GI detection primer having a base sequence S1 of at least 20 consecutive bases in the base sequence of positions 1 to 35 of SEQ ID NO: 1 or its complementary base sequence, or a base sequence S2 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence S1, and a second GI detection primer having a base sequence S3 of at least 20 consecutive bases in the base sequence of positions 60 to 95 of SEQ ID NO: 1 or its complementary base sequence, or a base sequence S4 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence S3, wherein the second GI detection primer is complementary to the DNA extension product of the first GI detection primer. [Item 24] The method according to Item 23, wherein the first GI detection primer has the base sequence S11 shown in either SEQ ID NO: 16 or 17 or its complementary base sequence S12, or a base sequence S21 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence S11 or S12, and the second GI detection primer has the base sequence S31 shown in either SEQ ID NO: 18 or 19 or its complementary base sequence S32, or a base sequence S41 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence S31 or S32. [Item 25] The method of any of Items 20 to 24, wherein the norovirus GII detection primer set used in the nucleic acid amplification reaction of step (2) comprises a first GII detection primer having a base sequence S5 of at least 20 consecutive bases in the base sequence of positions 1 to 50 of SEQ ID NO: 11 or a complementary base sequence thereof, or a base sequence S6 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence S5, and a second GII detection primer having a base sequence S7 of at least 18 consecutive bases in the base sequence of positions 80 to 107 of SEQ ID NO: 11 or a complementary base sequence thereof, or a base sequence S8 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence S7, wherein the second GII detection primer is complementary to a DNA extension product of the first GII detection primer. [Item 26] The method according to Item 25, wherein the first GII detection primer has the base sequence S51 shown in any one of SEQ ID NOs: 20 to 23 or its complementary base sequence S52, or the base sequence S61 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence S51 or S52, and the second GII detection primer has the base sequence S71 shown in any one of SEQ ID NOs: 24 and 25 or its complementary base sequence S72, or the base sequence S81 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence S71 or S72. [Item 27] ​​The method according to any one of Items 20 to 26, wherein the detecting step in step (3) is carried out by melting curve analysis. [Item 28] A kit for detecting GI and / or GII of norovirus in a single reaction, comprising the probe according to any one of Items 1 to 9 and the probe according to any one of Items 10 to 18. [Item 29] A kit for detecting GI and / or GII of norovirus in a single reaction, comprising the primer set according to Item 23 or 24 and the primer set according to Item 25 or 26. [Item 30] A kit for detecting GI and / or GII of norovirus in a single reaction, comprising the probe according to any one of Items 1 to 9, the probe according to any one of Items 10 to 18, the primer set according to Item 23 or 24, and the primer set according to Item 25 or 26. [Effects of the Invention]

[0010] The present invention can provide a useful method for detecting GI and / or GII of norovirus that may be contained in a sample. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows a representative example of the results of Test Example 1 (a graph showing the detection results when melting curve analysis was carried out using the probe shown in SEQ ID NO: 2). [Figure 2] FIG. 1 shows a representative example of the results of Test Example 2 (a graph showing the detection results when melting curve analysis was carried out using the probe shown in SEQ ID NO: 12). [Figure 3] FIG. 10 is a diagram showing a representative example of the results of Test Example 5 (a graph showing the detection results when melting curve analysis was performed using Combination No. A). [Figure 4] FIG. 1 shows a representative example of the results of Test Example 6 (a graph showing the detection results when melting curve analysis was performed using Combination No. A). [Figure 5] FIG. 1 shows the design of primers and probes for norovirus GI. [Figure 6] FIG. 1 shows the design of primers and probes for norovirus GII. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in more detail below by showing embodiments of the present invention, but the present invention is not limited to these. All non-patent and patent documents described in this specification are incorporated herein by reference in their entirety. In addition, the term "~" in this specification means "at least, at most," and for example, if the specification states "X~Y," it means "at least X and at most Y." In this specification, "and / or" means any one or any possible combination of two or more of the listed elements. In this specification, "comprising" encompasses the concepts of "consisting essentially of" and "consisting only of."

[0013] In this specification, a nucleic acid primer may be simply referred to as a primer, and a nucleic acid probe and a labeled probe may be simply referred to as a probe, and these are also collectively referred to as oligonucleotides.

[0014] In one embodiment, the present invention provides a method for detecting norovirus containing GI and / or GII using a labeled probe containing a specific base sequence. This method can, for example, detect norovirus with high sensitivity in a short period of time. In one embodiment, the present invention enables highly sensitive detection of norovirus GI and GII, for example, by melting curve analysis, by designing a labeled probe that targets a specific base sequence region derived from norovirus GI and a specific base sequence region derived from norovirus GII. Herein, SEQ ID NO: 1 is a base sequence derived from norovirus GI. SEQ ID NO: 11 is a base sequence derived from norovirus GII. The present invention encompasses labeled probes (also referred to herein as "nucleic acid probes") containing specific base sequences that target the specific regions of the base sequences shown in SEQ ID NOs: 1 and 11, respectively.

[0015] One embodiment of the present invention is a method for detecting the GI and / or GII of norovirus that may be contained in a sample (particularly for simultaneously detecting GI and GII in a single reaction). The method of the present invention uses a labeled probe having a specific base sequence, as described below. In a specific embodiment, the use of a single specific labeled probe in a reaction solution enables highly sensitive detection of the GI and / or GII of norovirus that may be contained in a sample in a single reaction, for example, by RT-PCR-melting curve analysis.

[0016] The sample is not particularly limited as long as it may contain the GI and / or GII of norovirus. Examples of samples include biological samples such as body fluids, excrement, and cells; and environmental samples such as wipes from facility walls, floors, equipment, fixtures, and toilets, or the rinsing fluid used to clean these. Examples of samples also include sputum, blood, urine, pus, cerebrospinal fluid, pleural effusion, ascites, gastric fluid, pharyngeal swabs, nasal swabs, saliva, oral scrapings, bronchial lavage fluid, alveolar lavage fluid, tissue sections, skin, vomit, and feces collected from subjects suspected of being infected with Mycobacterium tuberculosis complex, as well as isolated culture colonies and liquid culture fluids that may contain Mycobacterium tuberculosis complex. If necessary, each sample may be subjected to pretreatment or nucleic acid extraction, such as dilution or suspension, centrifugation, enzyme treatment, filtration, heat treatment, acid treatment, alkali treatment, organic solvent treatment, sonication, disruption, or grinding.

[0017] The method of collecting and preparing the sample is not particularly limited, and known methods can be used depending on the type and purpose of the sample.

[0018] The method for nucleic acid extraction is not particularly limited, and known methods can be used depending on the type of sample and the purpose. For nucleic acid extraction, for example, kits sold by various manufacturers may be used. Alternatively, an automatic extraction and purification device may be used.

[0019] In a specific embodiment, a method for detecting GI and / or GII of Norovirus that may be contained in a sample includes at least the following steps (1), (2), and (3): (1) reverse transcription (RT) of target RNA in a sample into cDNA; (2) generating one or more nucleic acid amplification products using the cDNA from step (1) as a template; and (3) detecting one or more nucleic acid amplification products of step (2) using one or more nucleic acid probes described below; Preferably, the method comprises the steps of: (1) performing step (2) by PCR reaction; and (3) performing step (3) by melting curve analysis (RT-PCR-melting curve analysis). Steps (1), (2), and (3) may be performed in the same reaction solution. Alternatively, two or more of steps (1), (2), and (3), for example, step (1) and step (2), may be performed consecutively or simultaneously.

[0020] [Process (1)] In one embodiment, step (1) preferably involves performing a reverse transcription reaction using a nucleic acid primer and a reverse transcriptase to generate cDNA from target RNA. The reverse transcriptase is not particularly limited as long as it has reverse transcription activity, and examples include reverse transcriptases (RNA-dependent DNA polymerases) derived from Moloney Murine Leukemia Virus (M-MLV) and Avian Myeloblastosis Virus (AMV), as well as mutants thereof. Examples of such mutants include those in which one to three amino acids are deleted, substituted, inserted, and / or added from the wild-type amino acid sequence, and those showing 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more amino acid sequence identity with the wild-type amino acid sequence. Specifically, examples include mutants lacking RNase H activity to increase the efficiency of cDNA synthesis. Furthermore, Tth DNA polymerase and its mutants are known to have reverse transcription activity under certain conditions, and can be used as reverse transcriptases in the present invention.

[0021] The nucleic acid primer used in the reverse transcription reaction may also serve as one of the primers used in the nucleic acid amplification reaction in step (2). The conditions for the reverse transcription reaction are not particularly limited as long as the reaction proceeds. The temperature for the reverse transcription reaction (or the temperature set in the reverse transcription reaction apparatus) is, for example, 37 to 55°C, preferably 40 to 52°C, and more preferably 42 to 50°C. The time for the reverse transcription reaction (or the time set in the reverse transcription reaction apparatus) is, for example, 0 seconds to 60 minutes, preferably 1 to 30 minutes, and more preferably 2 to 15 minutes.

[0022] [Process (2)] In one embodiment, step (2) is preferably a step of generating one or more nucleic acid amplification products by a nucleic acid amplification method (carrying out a nucleic acid amplification reaction using one or more nucleic acid primer sets). Nucleic acid amplification is a technique for amplifying a few copies of a target nucleic acid to a level at which it can be visualized, for example, hundreds of millions of copies or more, and is widely used not only in the field of life science research but also in fields such as clinical diagnosis, food hygiene testing, and environmental testing. Examples of such nucleic acid amplification methods include PCR, LAMP, LCR, TMA, SDA, RT-PCR, RT-LAMP, NASBA, RPA, TRC, and TMA. These techniques have already been established in the technical field, and a method can be selected according to the purpose. The nucleic acid amplification method is preferably PCR (including RT-PCR), but is not limited to this.

[0023] (PCR reaction) PCR is a reaction catalyzed primarily by DNA polymerase. PCR typically involves three steps: (i) DNA denaturation by heat treatment (dissociation of double-stranded DNA into single-stranded DNA), (ii) annealing of a primer to a single-stranded template DNA, and (iii) extension of the primer using a DNA polymerase, with each cycle being repeated. Examples of DNA polymerases include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, and DEEPVENT, as well as their variants. In the present invention, a DNA polymerase belonging to Family B is preferred for its simplicity, speed, high sensitivity, and resistance to sample-induced amplification inhibition. Furthermore, when step (3) is performed using melting curve analysis, a DNA polymerase belonging to Family B that lacks 5'->3' exonuclease activity is also preferred for use with a fluorescence-quenching probe.

[0024] The conditions for the PCR reaction are not particularly limited as long as the reaction proceeds. For example, the first step (i) may be performed at 80 to 100°C for approximately 0 to 300 seconds (e.g., approximately 0.5 to 300 seconds), and the second and subsequent (repeated) steps (i) may be performed at 80 to 100°C for approximately 0.5 to 300 seconds, step (ii) may be performed at 35 to 80°C for approximately 1 to 300 seconds, and step (iii) may be performed at 35 to 85°C for approximately 1 to 300 seconds. The cycle of steps (i) to (iii) is preferably repeated 30 to 70 times. The temperature and time of the repeated cycles may be changed every 1 to 3 cycles.

[0025] (DNA polymerase) The DNA polymerase that can be used in step (2) is preferably, but is not limited to, a DNA polymerase belonging to Family B. The DNA polymerase belonging to Family B is not particularly limited, but is preferably a DNA polymerase derived from Archea, more preferably a DNA polymerase derived from bacteria of the genera Pyrococcus and Thermococcus. Suitable DNA polymerases also include mutants of Family B archaea that do not lose their DNA polymerase activity. Examples of mutants include those in which one to three amino acids (e.g., one or two, or one) are deleted, substituted, inserted, and / or added to the wild-type amino acid sequence, or those that show 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more amino acid sequence identity with the wild-type amino acid sequence. Specifically, DNA polymerase mutants include, but are not limited to, those intended for enhancing polymerase activity, deleting exonuclease activity, adjusting substrate specificity, etc.

[0026] DNA polymerases derived from the genus Pyrococcus include, but are not limited to, DNA polymerases isolated from Pyrococcus furiosus, Pyrococcus sp. GB-D, Pyrococcus woesei, Pyrococcus abyssi, and Pyrococcus horikoshii, as well as mutants thereof derived therefrom that have not lost their DNA polymerase activity.

[0027] Examples of DNA polymerases derived from the genus Thermococcus include, but are not limited to, DNA polymerases isolated from Thermococcus kodakaraensis, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9°N-7 (Thermococcus sp. 9°N-7), and Thermococcus siculi, as well as mutants thereof that have not lost their DNA polymerase activity. DNA polymerases derived from Thermococcus kodakaraensis and mutants thereof (e.g., KOD-derived DNA polymerase lacking 3' to 5' exonuclease activity) are particularly suitable for use in the present invention due to their excellent extensibility and thermostability.

[0028] PCR enzymes using these DNA polymerases are commercially available, including Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England Biolabs), Tgo (Roche), and Pwo (Roche), any of which can be used in the present invention.

[0029] (KOD-derived DNA polymerase) As used herein, KOD-derived DNA polymerase (also referred to as KOD DNA polymerase) refers to a DNA polymerase derived from Thermococcus kodakaraensis and mutants thereof (e.g., a KOD-derived DNA polymerase in which 3' to 5' exonuclease activity has been deleted by substituting, deleting, inserting, and / or adding one to three (e.g., one, two, or one) amino acids in the naturally occurring amino acid sequence). In one preferred embodiment, step (2) involves performing a nucleic acid amplification reaction using such a KOD-derived DNA polymerase. Compared to Taq DNA polymerase, a Family A DNA polymerase, KOD DNA polymerase is superior in accuracy, amplification efficiency, extensibility, and resistance to amplification inhibition by sample-derived inhibitors. In the present invention, the use of such a KOD DNA polymerase is preferable in terms of simple, rapid, and highly sensitive detection of norovirus GI and / or GII, as shown in the Examples below.

[0030] (nucleic acid primer set) The nucleic acid primer set that can be used in step (2) is not particularly limited as long as it can amplify a norovirus-derived nucleic acid fragment that can form a complex with the probe described below. From the viewpoint of easily obtaining more sensitive determination results, the nucleic acid primer set is preferably a nucleic acid primer set that can amplify part or all of the nucleotide sequence shown in SEQ ID NO: 1 (e.g., the region from positions 10 to 85, preferably positions 15 to 80, more preferably positions 20 to 75, and even more preferably positions 25 to 70) for norovirus GI amplification, or a nucleic acid primer set that can amplify part or all of the nucleotide sequence shown in SEQ ID NO: 11 (e.g., the region from positions 30 to 95, preferably positions 35 to 90, more preferably positions 40 to 85, and even more preferably positions 40 to 80) for norovirus GII amplification.

[0031] For example, a nucleic acid primer set for detecting the GI of norovirus preferably includes a first GI detection primer having a base sequence S1 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence from positions 1 to 35 of SEQ ID NO: 1 or a complementary base sequence thereof, or a base sequence S2 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted, deleted, inserted, or added in the base sequence S1; and a second GI detection primer having a base sequence S3 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence from positions 60 to 95 of SEQ ID NO: 1 or a complementary base sequence thereof, or a base sequence S4 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted, deleted, inserted, or added in the base sequence S3, wherein the second GI detection primer is preferably a nucleic acid primer set complementary to the DNA extension product of the first GI detection primer. The base sequence S2 is preferably the base sequence S1 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted (particularly with mixed bases). The base sequence S4 is preferably the base sequence S3 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted (particularly with mixed bases).

[0032] An even more preferred nucleic acid primer set for GI detection comprises a first GI detection primer having the base sequence S11 shown in either SEQ ID NO: 16 or 17 or its complementary base sequence S12, or a base sequence S21 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted, deleted, inserted, or added in the base sequence S11 or S12, and a second GI detection primer having the base sequence S31 shown in either SEQ ID NO: 18 or 19 or its complementary base sequence S32, or a base sequence S41 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted, deleted, inserted, or added in the base sequence S31 or S32, wherein one GI detection primer is complementary to the DNA extension product of the other GI detection primer. From the viewpoint of facilitating highly sensitive detection with a high rate of fluorescence change even when detecting small amounts (e.g., approximately 1 to 20 copies) of norovirus, a nucleic acid primer set that can be used in the present invention is particularly preferably a nucleic acid primer set that includes: a first GI detection primer having the base sequence S13 of SEQ ID NO: 16 or its complementary base sequence S14, or a base sequence S22 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted, deleted, inserted, or added in the base sequence S13 or S14; and a second GI detection primer having the base sequence S33 of SEQ ID NO: 18 or its complementary base sequence S34, or a base sequence S42 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted, deleted, inserted, or added in the base sequence S33 or S34, wherein one GI detection primer is complementary to the DNA extension product of the other GI detection primer. The base sequence S21 is preferably the base sequence S11 or S12 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted (particularly with mixed bases).The base sequence S22 is preferably the base sequence S13 or S14 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted (particularly substituted with mixed bases). The base sequence S41 is preferably the base sequence S31 or S32 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted (particularly substituted with mixed bases). The base sequence S42 is preferably the base sequence S33 or S34 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted (particularly substituted with mixed bases).

[0033] Furthermore, the nucleic acid primer set for detecting norovirus GII preferably includes a first GII detection primer having a base sequence S5 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence from positions 1 to 50 of SEQ ID NO: 11 or a complementary base sequence thereof, or a base sequence S6 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted, deleted, inserted, or added in the base sequence S5; and a second GII detection primer having a base sequence S7 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence from positions 80 to 107 of SEQ ID NO: 11 or a complementary base sequence thereof, or a base sequence S8 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted, deleted, inserted, or added in the base sequence S7, wherein the second GII detection primer is complementary to a DNA extension product of the first GII detection primer. Base sequence S6 is preferably base sequence S5 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted (particularly with mixed bases). Base sequence S8 is preferably base sequence S7 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted (particularly with mixed bases).

[0034] An even more preferred nucleic acid primer set for detecting GII comprises a first GII detection primer having a base sequence S51 shown in any one of SEQ ID NOs: 20 to 23 or its complementary base sequence S52, or a base sequence S61 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted, deleted, inserted, or added in the base sequence S51 or S52, and a second GII detection primer having a base sequence S71 shown in any one of SEQ ID NOs: 24 and 25 or its complementary base sequence S72, or a base sequence S81 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted, deleted, inserted, or added in the base sequence S71 or S72, wherein one GII detection primer is complementary to the DNA extension product of the other GII detection primer. From the viewpoint of facilitating highly sensitive detection with a high rate of fluorescence change even when detecting small amounts (e.g., approximately 1 to 20 copies) of norovirus, a nucleic acid primer set that can be used in the present invention is particularly preferably a nucleic acid primer set that includes: a first GII detection primer having the nucleotide sequence S53 shown in any of SEQ ID NOs: 21 to 23 or its complementary nucleotide sequence S54, or the nucleotide sequence S62 obtained by substituting, deleting, inserting, or adding 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) nucleotides in the nucleotide sequence S53 or S54; and a second GII detection primer having the nucleotide sequence S73 shown in SEQ ID NO: 24 or its complementary nucleotide sequence S74, or the nucleotide sequence S82 obtained by substituting, deleting, inserting, or adding 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) nucleotides in the nucleotide sequence S73 or S74, wherein one GII detection primer is complementary to the DNA extension product of the other GII detection primer. The base sequence S61 is preferably the base sequence S51 or S52 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted (particularly with mixed bases).It is preferable that base sequence S62 is base sequence S53 or S54 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted (particularly substituted with mixed bases). It is preferable that base sequence S81 is base sequence S71 or S72 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted (particularly substituted with mixed bases). It is preferable that base sequence S82 is base sequence S73 or S74 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted (particularly substituted with mixed bases).

[0035] [Process (3)] Step (3) can be performed by any method known in the art. The target norovirus, like other infectious microorganisms, can mutate. Mismatches between the base sequence of a primer or probe and the base sequence of a target norovirus mutant can reduce the binding strength of the primer or probe to the target gene or its derived nucleic acid amplification product (target nucleic acid). In particular, in real-time PCR, if there are many mismatches between the target nucleic acid and the probe, the probe cannot sufficiently bind to the target nucleic acid, resulting in a delayed or complete absence of an amplification curve rise, which can lead to false negatives. In melting curve analysis, step (3) is performed after PCR is completed. Therefore, even if there are mismatches in the primer or probe, detection is possible as long as the final nucleic acid amplification product is obtained. Therefore, the impact of mismatches is less significant than in real-time PCR. Therefore, it is particularly preferable to detect the nucleic acid amplification product using melting curve analysis in step (3). Furthermore, by detecting nucleic acid amplification products using melting curve analysis, it is possible to detect them in a shorter time (for example, 45 minutes or less, preferably 40 minutes or less, and more preferably 35 minutes or less from the start of the nucleic acid amplification reaction to the completion of melting curve analysis).

[0036] In one embodiment, step (3) comprises the following steps (3-1) and (3-2): (3-1) hybridizing one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) with one or more nucleic acid probes of the present invention to form a complex; and (3-2) A step of detecting the complex of step (3-1) In order to obtain highly sensitive determination results in the detection of GI and / or GII of norovirus, it is preferable to use a nucleic acid probe described below that can specifically react with the nucleic acid amplification product that can be generated in the nucleic acid amplification reaction of step (2) to form a complex. The hybridization in step (3-1) is preferably carried out under temperature conditions that allow sufficient hybridization of the nucleic acid amplification product with the nucleic acid probe, such as, but not limited to, a temperature that is at least 5°C lower, more preferably at least 10°C lower than the Tm value of the nucleic acid probe.

[0037] (nucleic acid probe) The nucleic acid probe of the present invention is not particularly limited as long as it has the characteristics (A) and (B) or (C) and (D). (A) The nucleic acid has a base sequence A1 of at least 15 consecutive bases in the base sequence of positions 10 to 85 (preferably positions 15 to 80, more preferably positions 20 to 75, and even more preferably positions 25 to 70) of SEQ ID NO: 1 or its complementary base sequence, or a base sequence A2 in which 1 to 7 bases have been substituted, deleted, inserted or added in base sequence A1. (B) Only either the 5' or 3' end is labeled. (C) A base sequence C1 of at least 15 consecutive bases in the base sequence of positions 30 to 95 (preferably positions 35 to 90, more preferably positions 40 to 85, and even more preferably positions 40 to 80) of SEQ ID NO: 11 or its complementary base sequence, or a base sequence C2 in which 1 to 7 bases have been substituted, deleted, inserted or added in base sequence C1. (D) Only either the 5' or 3' end is labeled. When a nucleic acid probe is labeled with a fluorescence quenching dye that is quenched by interaction with guanine, as described below, it is preferable that at least one terminal base labeled with the dye is cytosine.

[0038] In one embodiment, the nucleic acid probe of the present invention can detect the GI of a norovirus having a base sequence that shows 70% or more, preferably 75% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, and particularly preferably 95% or more identity to the base sequence of positions 10 to 85 (preferably positions 15 to 80, more preferably positions 20 to 75, and even more preferably positions 25 to 70) of SEQ ID NO: 1 (including the norovirus genotype GI.1 (GenBank accession No. M87661) having the base sequence set forth in SEQ ID NO: 1 and its mutants). Examples of the mutants include GI.2 (GenBank accession no. L07418), GI.3 (GenBank accession no. U04469), GI.4 (GenBank accession no. AB042808), GI.5 (GenBank accession no. AJ277614), GI.6 (GenBank accession no. AF093797), GI.7 (GenBank accession no. AJ277609), GI.8 (GenBank accession no. AF538679), and GI.9 (GenBank accession no. HQ637267).

[0039] In one embodiment, the nucleic acid probe of the present invention can detect norovirus GII (including norovirus genotype GII.1 (GenBank accession No. U07611) having the base sequence set forth in SEQ ID NO: 11 and its mutants) that has a base sequence that shows 70% or more, preferably 75% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, and particularly preferably 95% or more identity to the base sequence of bases 30 to 95 (preferably bases 35 to 90, more preferably bases 40 to 85, and even more preferably bases 40 to 80) of SEQ ID NO: 11. The mutants include GII.2 (GenBank accession No. X81879), GII.3 (GenBank accession No. U02030), GII.4 (GenBank accession No. AJ277608), GII.8 (GenBank accession No. AF195848), GII.9 (GenBank accession No. AY038599), GII.10 (GenBank accession No. AF427118), GII.11 (GenBank accession No. AB074893), GII.12 (GenBank accession No. AJ277618), GII.13 (GenBank accession No. AY113106), GII.14(GenBank accession No. AY130761), GII.16(GenBank accession No. AY502010), GII.17(GenBank accession No. AY502009), GII.18(GenBank accession No. AY823304), GII.19(GenBank accession No. AY823306), GII.20 (GenBank accession No. EU373815), GII.21 (GenBank accession No.AY675554), GII.22 (GenBank accession no. AB083780), GII.23 (GenBank accession no. KT290889), GII.24 (GenBank accession no. KY225989), GII.25 (GenBank accession no. GQ856469), GII.26 (GenBank accession no. KU306738), GII.27 (GenBank accession no. MG495077), GII.NA1 (GenBank accession no. MG495079), and GII.NA2 (GenBank accession no. MG706448).

[0040] In certain embodiments, the nucleic acid probe of the present invention may be a probe containing a base sequence represented by (A) or (C) in which 1 to 7 bases have been substituted, deleted, inserted, or added (e.g., a probe for detecting the above-mentioned mutants), since noroviruses are prone to mutation and numerous subtypes exist. As used herein, the term "substitution" is used to encompass the substitution of a DNA base with an artificial nucleic acid base such as an LNA base (e.g., T → LNA-T). The number of bases that may be substituted, deleted, inserted, or added may be preferably 1 to 6, and more preferably 1 to 5. When a probe contains such base substitutions, deletions, insertions, or additions, it can also be said that the probe contains mismatched bases. It has been confirmed in the Examples described below that probes containing such mismatched bases can also be used suitably in the present invention.

[0041] As used herein, "containing a mismatched base" (or simply "mismatch") refers to containing a base that is not complementary to the base sequence of the target nucleic acid (when the target nucleic acid becomes double-stranded after a nucleic acid amplification reaction, the base sequence of one of the single-stranded nucleic acids resulting from the dissociation of the double strand). For example, when a cytosine base is present in the base sequence of the target nucleic acid, the base at the position corresponding to the cytosine base in the probe is a base other than guanine (e.g., an adenine base, a cytosine base, a thymine base, a universal base, a mixed base, an artificial nucleic acid base such as an LNA base). For example, when detecting a region of a target nucleic acid that is susceptible to mutation, a mismatched base (e.g., a universal base, a mixed base) can be selected at the position of the probe corresponding to the susceptible base. In one embodiment, the mismatched base is preferably a universal base and / or an artificial nucleic acid base.

[0042] The universal base refers to a base that can form a base pair with any of the four nucleic acid bases, i.e., adenine, cytosine, guanine, and thymine, or that does not form a base pair with any of the four nucleic acid bases. Any base that has the above-mentioned ability can be used as the universal base, and is preferably inosine, deoxyinosine, or 5-nitroindole, and more preferably inosine and / or deoxyinosine.

[0043] The mixed bases refer to a plurality of bases at a specific site constituting an oligonucleotide. Mixed bases are designated by a single letter of the alphabet by IUPAC, with A or T being represented as W, A or G as R, A or C as M, T or G as K, T or C as Y, G or C as S, A or C or T as H, G or C or T as B, A or G or C as V, A or G or T as D, and when all of A, T, G, and C are possible, they are represented as N. This specification also follows this notation.

[0044] From the viewpoint of enabling more sensitive detection, the nucleic acid probe of the present invention is preferably a fluorescently labeled probe having a base sequence A3 shown in any of SEQ ID NOs: 2 to 10 or its complementary base sequence A4, or a base sequence A5 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence A3 or A4, and / or a fluorescently labeled probe having a base sequence C3 shown in any of SEQ ID NOs: 12 to 15 or its complementary base sequence C4, or a base sequence C5 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence C3 or C4. Furthermore, from the viewpoint of enabling synergistically higher detection levels when used in combination with a fluorescently labeled probe having base sequence C6 shown in any of SEQ ID NOS: 12 and 13 or its complementary base sequence C7, or base sequence C9 obtained by substituting, deleting, inserting, or adding 1 to 5 bases in base sequence C7 or C8, a fluorescently labeled probe having base sequence A7 shown in any of SEQ ID NOS: 2 to 3 or its complementary base sequence A8, or base sequence A9 obtained by substituting, deleting, inserting, or adding 1 to 5 bases in base sequence A7 or A8 is preferred, and a fluorescently labeled probe having base sequence A9 shown in any of SEQ ID NOS: 2 and 3 or its complementary base sequence A10, or base sequence A11 obtained by substituting, deleting, inserting, or adding 1 to 5 bases in base sequence A9 or A10 is more preferred. SEQ ID NOS: A5 is preferably SEQ ID NOS: A3 or A4 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted (particularly with artificial nucleobases). SEQ ID NO: C5 is preferably SEQ ID NO: C3 or C4 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted (particularly with artificial nucleobases). SEQ ID NO: A9 is preferably SEQ ID NO: A7 or A8 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted (particularly with artificial nucleobases).It is preferable that SEQ ID NO: C9 is SEQ ID NO: C7 or C8 in which 1 to 5 (preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2) bases have been substituted (particularly with artificial nucleic acid bases).

[0045] The length of the nucleic acid probe of the present invention is not particularly limited as long as it is 15 bases or more, and is, for example, 16 bases or more, preferably 17 bases or more, and is usually 30 bases or less, preferably 25 bases or less, and more preferably 23 bases or less. The length of the nucleic acid probe of the present invention is more preferably 15 to 25 bases, and even more preferably 18 to 23 bases. In a specific embodiment, the length of the nucleic acid probe of the present invention may be 15 to 18 bases, 15 to 20 bases, 15 to 23 bases, 15 to 25 bases, or 15 to 30 bases. Use of a probe of such a length enables more sensitive detection of norovirus.

[0046] The above-mentioned probe is preferably labeled at only either the 5'-end or the 3'-end. In one embodiment, the nucleic acid probe of the present invention is preferably labeled so as to generate quenching or fluorescence when bound to a nucleic acid containing a base sequence that shows 70% or more, preferably 75% or more, more preferably 80% or more, even more preferably 90% or more, and even more preferably 95% or more identity to a base sequence complementary to the base sequence of the nucleic acid probe, and more preferably is labeled so as to generate quenching. The labeling substance is not particularly limited, but is more preferably a fluorescent dye.

[0047] The fluorescent dye may be either a fluorescent substance that emits fluorescence or a fluorescent substance that quenches fluorescence by hybridizing with a target nucleic acid amplification product to form a complex, but is preferably a fluorescent substance that quenches fluorescence when hybridized with a target nucleic acid amplification product, and is particularly preferably a fluorescence quenching dye that quenches fluorescence by interaction with guanine when hybridized with a target nucleic acid amplification product (for example, a fluorescence quenching dye that quenches fluorescence by interaction with guanine). Specific examples include, but are not limited to, at least one fluorescence quenching dye selected from the group consisting of fluorescein and its derivatives (e.g., fluorescein isothiocyanate (FITC)), rhodamine and its derivatives (e.g., 5-carboxyrhodamine 6G (GR6G), tetramethylrhodamine (TAMRA), carboxyrhodamine, x-rhodamine, sulforhodamine 101 acid chloride), and BODIPY and its derivatives (e.g., BODIPY-FL, BODIPY-FL / C3, BODIPY-FL / C6, BODIPY-5-FAM, BODIPY-TMR, BODIPY-TR, BODIPY-R6G, BODIPY-564, BODIPY-581, BODIPY-591, BODIPY-630, BODIPY-650, BODIPY-665).

[0048] More specifically, examples of fluorescence quenching dyes that are quenched by interaction with guanine include at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G (CR6G), TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue), and these fluorescence quenching dyes can be suitably used in the present invention.

[0049] In a particularly preferred embodiment, a probe labeled with a fluorescence quenching dye and having a terminal base of cytosine is more preferred. When such a probe hybridizes to a nucleic acid amplification product, it can form a base pair with a guanine base in the nucleic acid amplification product and interact with it to quench the fluorescence, making it very easy to measure changes in the fluorescence intensity of the reaction solution.

[0050] When the probe hybridizes, even if the cytosine base of the probe and the guanine base in the nucleic acid amplification product do not form a base pair, the fluorescence can be quenched as long as the distance between these bases is close. For example, details are described in Japanese Patent No. 5354216, and this technology can also be referenced in the present invention. That is, when the probe hybridizes, quenching can be achieved if the guanine base in the nucleic acid amplification product is located within, for example, 1 to 3 bases of the cytosine base of the probe (the base that forms a base pair with the cytosine base is counted as 1).

[0051] Therefore, even if at least one terminal base labeled with a fluorescence quenching dye is not cytosine, the change in fluorescence intensity of the reaction solution can be measured. For example, details are described in Japanese Patent No. 5354216, and this technology can also be used in the present invention. For example, when the probe hybridizes, quenching can be achieved if a guanine base in the nucleic acid amplification product is present within a range of, for example, 1 to 3 bases of the terminal base labeled with the fluorescence quenching dye (the base that forms a base pair with the terminal base is counted as 1).

[0052] In a particularly preferred embodiment, the nucleic acid probe of the present invention is used in step (3). Thus, the GI and / or GII of norovirus can be detected by using the nucleic acid probe of the present invention. The method of the present invention may use one type of probe of the present invention or a combination of two or more types of probes of the present invention.

[0053] In one embodiment, step (3) may include at least one of the following steps (3-a), (3-b), and (3-c): (3-a) Simultaneously with step (2), a step of hybridizing the nucleic acid probe of the present invention to the nucleic acid amplification product in the reaction solution and measuring the fluorescence intensity of the reaction solution to monitor the progress of the reaction (nucleic acid amplification reaction) in step (2) in real time. (3-b) After completion of step (2), a step of hybridizing the nucleic acid probe of the present invention to the nucleic acid amplification product in the reaction solution and measuring the fluorescence intensity of the reaction solution to monitor the progress of the reaction (nucleic acid amplification reaction) in step (2) at an endpoint. (3-c) After step (2), hybridizing the nucleic acid probe of the present invention to the nucleic acid amplification product in the reaction solution and measuring the temperature dependence of the fluorescence intensity of the reaction solution. Step (3-a), (3-b), or (3-c) allows for simple, rapid, and highly sensitive detection of the formation of a complex between a nucleic acid amplification product and a nucleic acid probe. Steps (3-a), (3-b), and (3-c) may be performed in combination; for example, both steps (3-a) and (3-b) or both steps (3-a) and (3-c) may be performed. In one embodiment, step (3-b) or (3-c) is preferred from the viewpoint of more rapid detection of a nucleic acid amplification product. Step (3-c), i.e., melting curve analysis, is particularly preferred.

[0054] (Step (3-a)) Step (3-a) is a method for monitoring the progress of the nucleic acid amplification reaction in real time (so-called real-time PCR method), and quantitative analysis is possible by comparing with a control substance of known concentration.

[0055] (Step (3-b)) In step (3-b), the progress of the nucleic acid amplification reaction is monitored at the endpoint, allowing rapid detection of the target nucleic acid contained in the sample. Furthermore, the approximate amount of target nucleic acid can be estimated by comparing the fluorescence intensity at the endpoint. For example, the progress of a nucleic acid amplification reaction is monitored at an endpoint by measuring the fluorescence intensity of a reaction solution containing a nucleic acid probe labeled with a fluorescence quenching dye. After the nucleic acid amplification reaction is completed, the fluorescence intensity of the reaction solution is measured and compared with the fluorescence intensity of the reaction solution before the reaction, thereby confirming whether or not the target nucleic acid has been amplified. Alternatively, the presence or absence of the target nucleic acid in a sample can be confirmed by comparing the fluorescence intensity of the reaction solution after the reaction with the fluorescence intensity of a control reaction solution. A control reaction solution is a reaction solution to which a sample known to be negative or positive has been added instead of the sample to be measured. The progress of the nucleic acid amplification reaction generally needs to be monitored in real time, but for the purpose of more rapid and simple detection, it is preferable to measure at the end point.

[0056] (Step (3-c)) In step (3-c), measuring the temperature dependence of fluorescence intensity can specifically mean measuring the fluorescence intensity at each temperature while changing the temperature of the reaction solution from low to high. The melting temperature (Tm value) specific to the nucleic acid probe used can be determined by first differentiating the obtained fluorescence intensity with respect to temperature. Furthermore, the fluorescence intensity may be converted into a fluorescence quenching rate or the like depending on the purpose. The detection and analysis of target nucleic acids using the Tm value is called melting curve analysis. Generally, the Tm value refers to the temperature at which the proportion of an oligonucleotide that forms a double strand with its complementary strand is equal to the proportion that remains single-stranded. Because the Tm value is a value specific to a base sequence, melting curve analysis can be used as a method for analyzing base sequence polymorphisms in target nucleic acids. Base sequence polymorphisms referred to here include single nucleotide polymorphisms, base substitutions, base deletions, base insertions, etc.

[0057] For example, melting curve analysis is also used in SNP analysis. If there is a mutation in the base sequence of the target nucleic acid relative to the probe, the bases will mismatch when the probe hybridizes, and the Tm value will generally be low. Therefore, single nucleotide polymorphism analysis (SNP analysis) can also be performed by comparing the magnitude of the Tm value.

[0058] [Reagents for detecting GI and / or GII of norovirus] In another embodiment, the present invention provides a reagent for detecting norovirus. The reagent preferably contains at least components necessary for nucleic acid amplification and detection, in addition to the nucleic acid probe of the present invention (a norovirus detection probe containing GI and / or GII) described above. These necessary components can be any known components. For example, the reagent of the present invention preferably contains at least a nucleic acid primer set (e.g., a nucleic acid primer set for PCR), DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and inorganic salts such as magnesium salts. Multiple sets of nucleic acid primer sets and detection nucleic acid probes can be included to amplify multiple regions of norovirus, including GI and / or GII. The concentrations of each component can be adjusted as appropriate; for example, the nucleic acid probe is preferably 0.01 to 1 μM, and more preferably 0.02 to 0.5 μM. When used as a nucleic acid probe set, each nucleic acid probe contained in the probe set is preferably within the above-mentioned concentration range. The nucleic acid primer is preferably 0.01 to 10 μM. When used as a nucleic acid primer set, each nucleic acid primer contained in the primer set is preferably within the above-mentioned concentration range. DNA polymerase is preferably 0.01 to 1 U / μL, more preferably 0.02 to 0.5 U / μL. Deoxyribonucleoside triphosphates (dNTPs) are preferably 0.02 to 1 mM, more preferably 0.1 to 0.5 mM. Inorganic salts such as magnesium salts are preferably 0.1 to 10 mM, more preferably 1 to 5 mM.

[0059] Furthermore, the reagent of the present invention may contain additives known in the art for the purposes of suppressing nonspecific amplification or promoting the reaction. Examples of additives for suppressing nonspecific amplification include known anti-DNA polymerase antibodies and phosphate. Examples of additives for promoting the reaction include bovine serum albumin (BSA), protease inhibitors, single-strand binding protein (SSB), T4 gene 32 protein, tRNA, sulfur- or acetic acid-containing compounds, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine, ectoine, trehalose, dextran, polyvinylpyrrolidone (PVP), gelatin, tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), polyethylene glycol, carnitine, Triton, Tween 20, and Nonidet® P40. In addition, to facilitate the determination of false negatives, the reagent of the present invention preferably contains an internal control known in the art. In the present invention, these additives may be used alone or in combination of two or more.

[0060] In certain embodiments, the reagent of the present invention preferably includes an internal control. By using an internal control in combination, it is possible to easily confirm that the nucleic acid amplification reaction is proceeding normally, reducing the risk of false negatives and enabling more accurate norovirus testing.

[0061] [Kit for detecting GI and / or GII of norovirus] In another embodiment, the present invention provides a kit for detecting norovirus. The kit of the present invention is not particularly limited as long as it contains the nucleic acid probe of the present invention (a probe for detecting norovirus GI and / or a probe for detecting norovirus GII) or the reagent of the present invention described above and is configured to detect (including differentiate) norovirus. For example, the kit of the present invention can optionally contain a reagent capable of detecting (including quantitating) the presence of the target substance and / or instructions for use, etc. For example, the kit of the present invention can be provided in a form in which the nucleic acid probe, components necessary for the nucleic acid amplification reaction, and components necessary for detecting the amplification product are sealed in the same container or in separate containers, packaged in a single package, and information on how to use the kit is included. The kit of the present invention can also contain a positive control solution and / or a negative control solution. [Example]

[0062] The present invention will be specifically explained below by showing examples, but the present invention is not limited to these examples.

[0063] Test Example 1: Confirmatory test for GI detection of norovirus (1-1) Method To search for a nucleic acid probe capable of specifically detecting norovirus, 50 copies of norovirus GI RNA were used per reaction, and the presence or absence of nonspecific amplification and detection of norovirus were confirmed using a total of nine nucleic acid probes designed based on the base sequence shown in SEQ ID NO: 1. Purified water was used as a negative sample for the presence or absence of nonspecific amplification. The nucleic acid probes used were oligonucleotides consisting of the base sequences shown in any of SEQ ID NOs: 2 to 10, synthesized according to standard methods (labeled with BODIPY-FL at either the 5' or 3' end; see Figure 5 for the labeling position in each probe). The nucleic acid primer set used consisted of a nucleic acid primer consisting of the base sequence shown in SEQ ID NO: 16 and a nucleic acid primer consisting of the base sequence shown in SEQ ID NO: 18.

[0064] (1-2) Reaction solution The following solutions were prepared using GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace®. GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace® were used in the amounts adjusted according to their respective instruction manuals (ReverTra Ace® was used at 0.1 U / μL). An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. 1.5 μM primer shown in SEQ ID NO: 16 4.5 μM primer shown in SEQ ID NO: 18 0.3 μM of a probe represented by any one of SEQ ID NOs: 2 to 10 (The probes represented by SEQ ID NOs: 2 to 4 have the 7th and 20th bases, respectively.) It is substituted with LNA bases.) (1-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (reverse transcription, nucleic acid amplification, and melting curve analysis) 42℃・2 minutes 97℃・15 seconds (1 cycle) 97℃・1 second 55℃・3 seconds 68℃・3 seconds (more than 60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.4℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes. (1-4) Results Figure 1 shows a detection graph obtained when 50 copies of norovirus GI were detected by nucleic acid amplification and melting curve analysis using the probe shown in SEQ ID NO: 2 as a representative example. Similarly, the measurement results using the probe used in this test example are summarized in Table 1. The rate of change in fluorescence (representing the quenching rate in this test example) was calculated using the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0065] As shown in the results in Table 1, even when probes were designed in the same region, high fluorescence intensity and stable detection were possible depending on the probe sequence. Furthermore, when nonspecific amplification was confirmed using purified water as a negative sample, no nonspecific amplification was observed regardless of the probe used.

[0066] [Table 1]

[0067] Test Example 2: Confirmatory test for detection of norovirus GII (2-1) Method To search for a nucleic acid probe capable of specifically detecting norovirus, 50 copies of norovirus RNA were used per reaction, and the presence or absence of nonspecific amplification and detection of norovirus were confirmed using a total of four nucleic acid probes designed based on the base sequence shown in SEQ ID NO: 11. Purified water was used as a negative sample for the measurement to check for the presence or absence of nonspecific amplification. The nucleic acid probes used were oligonucleotides consisting of the base sequences shown in any of SEQ ID NOs: 12 to 15, synthesized according to standard methods (only either the 5' or 3' end was labeled with CR6G; see Figure 6 for the labeling position in each probe). The nucleic acid primer set used consisted of a nucleic acid primer consisting of the base sequence shown in SEQ ID NO: 22 and a nucleic acid primer consisting of the base sequence shown in SEQ ID NO: 24.

[0068] (2-2) Reaction solution The following solutions were prepared using GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace®. GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace® were used in the amounts adjusted according to their respective instruction manuals (ReverTra Ace® was used at 0.1 U / μL). An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. 1.0 μM primer shown in SEQ ID NO: 22 0.5 μM primer shown in SEQ ID NO: 23 0.1 μM primer shown in SEQ ID NO: 21 4.5 μM Primer shown in any one of SEQ ID NOs: 24 0.3 μM of a probe represented by any one of SEQ ID NOs: 12 to 15 (2-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (reverse transcription, nucleic acid amplification, and melting curve analysis) 42℃・2 minutes 97℃・15 seconds (1 cycle) 97℃・1 second 55℃・3 seconds 68℃・3 seconds (more than 60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.4℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes. (2-4) Results Figure 2 shows a detection graph obtained when 50 copies of norovirus GII were detected by nucleic acid amplification and melting curve analysis using the probe shown in SEQ ID NO: 12 as a representative example. Similarly, the measurement results using the probe used in this test example are summarized in Table 2. The rate of change in fluorescence (representing the quenching rate in this test example) was calculated using the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0069] As shown in the results in Table 2, even when probes were designed in the same region, high fluorescence intensity and stable detection were possible depending on the probe sequence. Furthermore, when nonspecific amplification was confirmed using purified water as a negative sample, no nonspecific amplification was observed regardless of the probe used.

[0070] [Table 2]

[0071] Test Example 3: Detection confirmation test using nucleic acid primer combinations for norovirus GI (3-1) Method Based on the results of Test Example 1, the combination of nucleic acid primers for detecting norovirus GI was verified. Fifty copies of norovirus RNA were used per reaction, and the presence or absence of nonspecific amplification and the detection of norovirus were confirmed using a total of four nucleic acid primer combinations designed based on the base sequence shown in SEQ ID NO: 1. Purified water was used as a negative sample for the measurement of the presence or absence of nonspecific amplification. The nucleic acid probe used was an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2, synthesized according to a standard method (only either the 5' or 3' end was labeled with BODIPY-FL. See Figure 5 for the labeling position in each probe). The nucleic acid primer set used consisted of a nucleic acid primer consisting of a base sequence shown in any one of SEQ ID NOs: 16 to 17 and a set of nucleic acid primers consisting of a base sequence shown in any one of SEQ ID NOs: 18 to 19.

[0072] (3-2) Reaction solution The following solutions were prepared using GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace®. GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace® were used in the amounts adjusted according to their respective instruction manuals (ReverTra Ace® was used at 0.1 U / μL). An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. 1.5 μM Primer shown in either SEQ ID NO: 16 or 17 4.5 μM Primer shown in either SEQ ID NO: 18 or 19 0.3 μM probe shown in SEQ ID NO: 2 (3-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (reverse transcription, nucleic acid amplification, and melting curve analysis) 42℃・2 minutes 97℃・15 seconds (1 cycle) 97℃・1 second 55℃・3 seconds 68℃・3 seconds (more than 60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.4℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes. (3-4) Results The measurement results for the combinations of nucleic acid primers used in this test example are summarized in Table 3. The rate of change in fluorescence (representing the quenching rate in this test example) was calculated using the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0073] As shown in the results in Table 3, even when nucleic acid primers were designed in the same region, high fluorescence intensity and stable detection were possible depending on the nucleic acid primer sequence. Furthermore, when nonspecific amplification was confirmed using purified water as a negative sample, no nonspecific amplification was observed regardless of which primer was used.

[0074] [Table 3]

[0075] Test Example 4: Detection confirmation test using a combination of nucleic acid primers for norovirus GII (4-1) Method Based on the results of Test Example 2, the combination of nucleic acid primers for detecting norovirus GII was verified. Fifty copies of norovirus RNA were used per reaction, and the presence or absence of nonspecific amplification and the detection of norovirus were confirmed using a combination of four nucleic acid primers designed based on the base sequence shown in SEQ ID NO: 11. Purified water was used as a negative sample for the measurement of the presence or absence of nonspecific amplification. The nucleic acid probe used was an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 13, synthesized according to a standard method (only either the 5' or 3' end was labeled with CR6G; see Figure 6 for the labeling position in each probe). The nucleic acid primer set used consisted of a nucleic acid primer consisting of a base sequence shown in any of SEQ ID NOs: 20 to 23 and a set of nucleic acid primers consisting of a base sequence shown in any of SEQ ID NOs: 24 and 25.

[0076] (4-2) Reaction solution The following solutions were prepared using GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace®. GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace® were used in the amounts adjusted according to their respective instruction manuals (ReverTra Ace® was used at 0.1 U / μL). An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. 1.0 μM Primer shown in either SEQ ID NO: 20 or 22 0.5 μM primer shown in SEQ ID NO: 23 (only when SEQ ID NO: 22 is included) 0.1 μM primer shown in SEQ ID NO: 21 4.5 μM Primer shown in either SEQ ID NO: 24 or 25 0.3 μM probe shown in SEQ ID NO: 13 (4-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (reverse transcription, nucleic acid amplification, and melting curve analysis) 42℃・2 minutes 97℃・15 seconds (1 cycle) 97℃・1 second 55℃・3 seconds 68℃・3 seconds (more than 60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.4℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes. (4-4) Results The measurement results for the combinations of nucleic acid primers used in this test example are summarized in Table 4. The rate of change in fluorescence (representing the quenching rate in this test example) was calculated using the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0077] As shown in the results in Table 4, even when nucleic acid primers were designed in the same region, high fluorescence intensity and stable detection were possible depending on the nucleic acid primer sequence. Furthermore, when nonspecific amplification was confirmed using purified water as a negative sample, no nonspecific amplification was observed regardless of which probe was used.

[0078] [Table 4]

[0079] Test Example 5: Simultaneous detection confirmation test of GI and / or GII of norovirus (5-1) Method Based on the results obtained so far, we verified the simultaneous detection of norovirus GI and GII. We confirmed the presence or absence of nonspecific amplification and norovirus detection using a combination of a norovirus GI detection primer probe and a norovirus GII detection primer probe when using 50 copies or 10 copies of norovirus GI and GII RNA per reaction. Purified water was used as a negative sample for the nonspecific amplification test. The nucleic acid probes used were oligonucleotides consisting of the base sequence shown in SEQ ID NO: 2 for norovirus GI detection and either SEQ ID NO: 12 or 13 for norovirus GII detection, synthesized according to standard methods (labeled with BODIPY-FL at either the 5' or 3' end; see Figures 5 and 6 for the labeling positions in each probe).

[0080] (5-2) Reaction solution The following solutions were prepared using GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace®. GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace® were used in the amounts adjusted according to their respective instruction manuals (ReverTra Ace® was used at 0.1 U / μL). An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. For detecting norovirus GI 1.5 μM primer shown in SEQ ID NO: 16 4.5 μM Primer shown in either SEQ ID NO: 18 or 19 0.3 μM probes represented by any of SEQ ID NOs: 2, 4, and 5 Norovirus GII detection 1.0 μM Primer shown in either SEQ ID NO: 20 or 22 0.5 μM primer shown in SEQ ID NO: 23 (only when SEQ ID NO: 22 is included) 0.1 μM primer shown in SEQ ID NO: 21 4.5 μM Primer shown in either SEQ ID NO: 24 or 25 0.3 μM probes shown in any of SEQ ID NOs: 12, 13, and 15 (5-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (reverse transcription, nucleic acid amplification, and melting curve analysis) 42℃・2 minutes 97℃・15 seconds (1 cycle) 97℃・1 second 55℃・3 seconds 68℃・3 seconds (more than 60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.4℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes. (5-4) Results The measurement results for the combination of norovirus GI and GII used in this test example are summarized in Table 5. The fluorescence change rate (representing the quenching rate in this test example) was calculated using the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0081] Figure 3 shows a representative example of the detection graph obtained when GI and GII detection of 10 copies of norovirus was performed using combination No. A by nucleic acid amplification and melting curve analysis. As shown in Table 5, it was confirmed that 10 copies of norovirus could be detected with each combination. Furthermore, when nonspecific amplification was confirmed using purified water as a negative sample, no nonspecific amplification was observed with any of the probes used.

[0082] [Table 5]

[0083] Test Example 6: Simultaneous detection confirmation test of GI and / or GII of norovirus using fecal specimens (6-1) Method Based on the results obtained so far, we confirmed whether norovirus detection is possible using simple processing of fecal samples. Negative fecal samples were suspended in purified water and centrifuged at 13,000 rpm. The supernatant was then subjected to 50 copies of norovirus GI and GII RNA per reaction, and the detection of norovirus GI and GII was confirmed. The nucleic acid probes used for norovirus GI detection were oligonucleotides consisting of the base sequence shown in SEQ ID NO: 2 and SEQ ID NO: 12, synthesized according to standard methods (only either the 5' or 3' end was labeled with BODIPY-FL. See Figures 5 and 6 for the labeling positions in each probe).

[0084] (6-2) Reaction solution The following solutions were prepared using GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace®. GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace® were used in the amounts adjusted according to their respective instruction manuals (ReverTra Ace® was used at 0.1 U / μL). An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. (Combination No. A) For detecting norovirus GI 1.5 μM primer shown in SEQ ID NO: 16 4.5 μM primer shown in SEQ ID NO: 18 0.3 μM probe shown in SEQ ID NO: 2 Norovirus GII detection 1.0 μM primer shown in SEQ ID NO: 22 0.5 μM primer shown in SEQ ID NO: 23 0.1 μM primer shown in SEQ ID NO: 21 4.5 μM primer shown in SEQ ID NO: 24 0.3 μM probe shown in SEQ ID NO: 12 (6-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (reverse transcription, nucleic acid amplification, and melting curve analysis) 42℃・2 minutes 97℃・15 seconds (1 cycle) 97℃・1 second 55℃・3 seconds 68℃・3 seconds (more than 60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.4℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes. (6-4)Result Figure 4 shows a detection graph obtained when a stool sample suspended in purified water was centrifuged at 13,000 rpm and the supernatant was supplemented with norovirus GI and GII RNA at 50 copies / test. The supernatant from the negative stool sample used in this test did not undergo a nucleic acid purification process and therefore contained biological contaminants such as various proteins that inhibit nucleic acid amplification reactions. The results of this test confirmed that the present invention can be used to perform high-sensitivity assays without problems even on samples that inhibit nucleic acid amplification reactions, even when the stool sample was simply processed. [Industrial Applicability]

[0085] By using a method, reagent, or kit that uses the nucleic acid probe of the present invention, norovirus GI and GII that may be present in a sample can be detected easily and with high sensitivity. Therefore, the present invention is not only useful for research purposes, but also enables early detection and early treatment of norovirus GI and / or GII without being affected by the skill of the operator or the performance of the equipment, leading to the prevention of the spread of sexually transmitted diseases and making a significant contribution to clinical diagnosis, environmental testing, and the like.

Claims

1. A probe for detecting GI of norovirus, having the following characteristics (A) and (B): (A) It has a base sequence A1 of at least 15 consecutive bases in the base sequence from 10 to 85 of SEQ ID NO: 1 or its complementary base sequence, or a base sequence A2 in which 1 to 7 bases have been substituted, deleted, inserted or added in base sequence A1. (B) Only either the 5' or 3' end is labeled.

2. 2. The probe according to claim 1, wherein the length of the base sequence of (A) is 15 to 30 bases.

3. The probe according to claim 1, wherein the base sequence of (A) comprises a base sequence A3 represented by any one of SEQ ID NOs: 2 to 10, a complementary base sequence A4 thereto, or a base sequence A5 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence A3 or A4.

4. The probe according to claim 1 , wherein the label (B) is a fluorescent dye label.

5. The probe according to claim 1, wherein the label (B) is a label with a fluorescent quenching dye that is quenched when bound to a nucleic acid containing a base sequence that is 70% or more identical to a base sequence complementary to the base sequence of the probe.

6. The probe according to claim 1, wherein the label (B) is a label with a fluorescence quenching dye that is quenched by interaction with guanine.

7. The probe according to claim 1, wherein the label (B) is a label with at least one fluorescence quenching dye selected from the group consisting of fluorescein and its derivatives, rhodamine and its derivatives, and BODIPY and its derivatives.

8. The probe according to claim 1, wherein the label (B) is a label with at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue).

9. The probe according to claim 1 , wherein in (B), the labeled terminal base is cytosine.

10. A probe for detecting GII of norovirus, having the following characteristics (C) and (D): (C) A base sequence C1 of at least 15 consecutive bases in the base sequence from bases 30 to 95 of SEQ ID NO: 11 or its complementary base sequence, or a base sequence C2 in which 1 to 7 bases have been substituted, deleted, inserted or added in the base sequence C1. (D) Only either the 5' or 3' end is labeled.

11. The probe according to claim 10, wherein the length of the base sequence of (C) is 15 to 25 bases.

12. The probe according to claim 10, wherein the base sequence of (C) comprises a base sequence C3 represented by any one of SEQ ID NOs: 12 to 15 or a complementary base sequence C4 thereof, or a base sequence C5 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence C3 or C4.

13. The probe according to claim 10 , wherein the label (D) is a fluorescent dye label.

14. The probe according to claim 10, wherein the label (D) is a label with a fluorescence quenching dye that is quenched when bound to a nucleic acid containing a base sequence that shows 70% or more identity with a base sequence complementary to the base sequence of the probe.

15. The probe according to claim 10 , wherein the label (D) is a label with a fluorescence quenching dye that is quenched by interaction with guanine.

16. The probe according to claim 10, wherein the label (D) is a label with at least one fluorescence quenching dye selected from the group consisting of fluorescein and its derivatives, rhodamine and its derivatives, and BODIPY and its derivatives.

17. The probe according to claim 10, wherein the label (D) is a label with at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue).

18. The probe according to claim 10, wherein in (D), the labeled terminal base is cytosine.

19. A method for detecting GI and / or GII of norovirus that may be contained in a sample in a single reaction using a probe according to any one of claims 1 to 9 and a probe according to any one of claims 10 to 18.

20. The following steps (1), (2), and (3): (1) providing a sample that may contain GI and / or GII of norovirus; (2) performing a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1); and (3) detecting one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) using one or more probes according to any one of claims 1 to 9 and one or more probes according to any one of claims 10 to 18; 20. The method of claim 19, comprising:

21. The method according to claim 20, wherein the step (2) is carried out by PCR reaction, and the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to family B.

22. The method according to claim 21 , wherein the DNA polymerase belonging to family B is a DNA polymerase derived from KOD or a mutant thereof.

23. The method of claim 20, wherein the norovirus GI detection primer set used in the nucleic acid amplification reaction of step (2) comprises a first GI detection primer having a base sequence S1 of at least 20 consecutive bases in the base sequence of positions 1 to 35 of SEQ ID NO: 1 or a complementary base sequence thereof, or a base sequence S2 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence S1; and a second GI detection primer having a base sequence S3 of at least 20 consecutive bases in the base sequence of positions 60 to 95 of SEQ ID NO: 1 or a complementary base sequence thereof, or a base sequence S4 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence S3, wherein the second GI detection primer is complementary to a DNA extension product of the first GI detection primer.

24. The method according to claim 23, wherein the first GI detection primer has a base sequence S11 shown in either SEQ ID NO: 16 or 17 or a complementary base sequence S12 thereof, or a base sequence S21 obtained by substituting, deleting, inserting or adding 1 to 5 bases in the base sequence S11 or S12, and the second GI detection primer has a base sequence S31 shown in either SEQ ID NO: 18 or 19 or a complementary base sequence S32 thereof, or a base sequence S41 obtained by substituting, deleting, inserting or adding 1 to 5 bases in the base sequence S31 or S32.

25. The method of claim 20, wherein the norovirus GII detection primer set used in the nucleic acid amplification reaction of step (2) comprises a first GII detection primer having a base sequence S5 of at least 20 consecutive bases in the base sequence from positions 1 to 50 of SEQ ID NO: 11 or a complementary base sequence thereof, or a base sequence S6 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence S5, and a second GII detection primer having a base sequence S7 of at least 18 consecutive bases in the base sequence from positions 80 to 107 of SEQ ID NO: 11 or a complementary base sequence thereof, or a base sequence S8 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence S7, wherein the second GII detection primer is complementary to a DNA extension product of the first GII detection primer.

26. The method according to claim 25, wherein the first GII detection primer has a base sequence S51 shown in any one of SEQ ID NOs: 20 to 23 or a complementary base sequence S52 thereof, or a base sequence S61 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence S51 or S52, and the second GII detection primer has a base sequence S71 shown in any one of SEQ ID NOs: 24 and 25 or a complementary base sequence S72 thereof, or a base sequence S81 in which 1 to 5 bases have been substituted, deleted, inserted or added in the base sequence S71 or S72.

27. 21. The method of claim 20, wherein the detecting step in step (3) is performed by melting curve analysis.

28. A kit for detecting GI and GII of norovirus in one reaction, comprising the probe according to any one of claims 1 to 9 and the probe according to any one of claims 10 to 18.

29. A kit for detecting noroviruses including GI and GII in one reaction, comprising the primer set according to claim 23 or 24 and the primer set according to claim 25 or 26.

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