Oligonucleotides for detecting norovirus

Abstract

To provide a means for highly sensitive detection of the presence of GII type norovirus in a sample.SOLUTION: The present invention provides a method for testing the presence of GII type norovirus in a sample and comprises the steps of: (1) preparing a pair of nucleic acid primer sets of a combination of nucleic acid primers consisting of a specific base sequence capable of detecting GII type norovirus; (2) preparing a mixed solution in which a sample and a 1-step RT-PCR reaction solution containing a pair of nucleic acid primer sets and a thermostable DNA polymerase prepared in the above step (1) are mixed; and (3) carrying out a 1-step RT-PCR reaction after sealing a reaction vessel.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an oligonucleotide that can be used as a nucleic acid primer for detecting GII type norovirus contained in a sample. The present invention further relates to a method for detecting GII type norovirus contained in a sample using the oligonucleotide and a kit therefor. The present invention makes it possible to detect norovirus in, for example, fecal samples, blood samples, environmental swabs, etc. with high sensitivity. The present invention can be used in life science research, clinical diagnosis, food hygiene testing, environmental testing, etc. [Background technology]

[0002] Norovirus is a virus that causes food poisoning, and it is said that half of the food poisoning cases in Japan are caused by norovirus (Non-Patent Document 1). Therefore, it goes without saying that simple, rapid, and highly sensitive detection of norovirus is important in clinical diagnosis, food hygiene inspection, environmental inspection, and the like.

[0003] For pathogen testing of norovirus, a tissue culture method has not been established, and instead, electron microscopy, immunological antigen detection by ELISA, or detection methods for viral genes using nucleic acid amplification technology have been developed. Among these testing methods, nucleic acid amplification technology, which can detect norovirus with high sensitivity, is widely used. Since the base sequence of the RNA genome of norovirus is highly diverse, several techniques have been developed for detection by nucleic acid amplification (e.g., Patent Document 1, Patent Document 2, Patent Document 3, Patent Document 4, Patent Document 5). Among these, in Japan, the real-time reverse transcription polymerase chain reaction (RT-PCR) method based on a notice from the Surveillance and Safety Division, Safety Department, Pharmaceuticals and Food Safety Bureau, Ministry of Health, Labor and Welfare (Food Safety Inspection No. 1105001) is widely used as the official method.

[0004] The main cause of norovirus infection is eating food contaminated with the virus. In addition, since many cases of infection are transmitted through human hands, regular stool tests are required in food preparation facilities, medical facilities, elderly care facilities, and daycare centers. The Mass Food Preparation Facility Sanitation Management Manual adds that regular stool tests for food preparation staff should include norovirus tests at least once a month or as necessary during the norovirus epidemic period from October to March.

[0005] In addition to regular stool tests, swab tests are useful for clarifying the route of contamination and grasping the contamination status of facility environments. Examples of swab tests include swabs and other utensils such as cutting boards, knives, dishcloths, and tableware, refrigerator handles, bathroom doorknobs, faucets in washrooms, kitchens, toilets, and bathrooms, cooks' hands and fingers, bathrooms, washbasins, handrails, and rooms, and other facilities, which are then eluted in water or a buffer solution. Stool and swab test samples contain a large amount of feces and dirt and dust from the testing site, and therefore contain a large amount of contaminants that inhibit RT-PCR and nucleic acids other than those derived from norovirus that cause nonspecific amplification.

[0006] Nucleic acid primers are one of the important elements for amplifying specific genes from samples containing many impurities using the RT-PCR method and detecting them with high sensitivity. As an example of a primer pair, primers for detecting norovirus include the primers (SEQ ID NOs: 1 to 5) described in the notification (Food Safety Inspection No. 1105001) of the Surveillance and Safety Division, Safety Department, Pharmaceuticals and Food Safety Bureau, Ministry of Health, Labor and Welfare, and are widely used.

[0007] Generally, when multiple types of nucleic acids are simultaneously amplified and detected using a one-step RT-PCR reaction, multiple types of nucleic acid primer sets are included in one RT-PCR reaction system. In this case, the reverse transcription reaction and PCR reaction for multiple types of target nucleic acids are performed in one solution, so it is known that the multiple types of primer sets included in the reaction solution mutually affect the amplification reaction of each nucleic acid. The fact that the Tm values ​​of each primer are different also affects the reaction system. As a result, non-specific amplification or fundamental amplification failure occurs in each of the reverse transcription reaction and PCR reaction. For this reason, the selection of the nucleic acid primer set in the one-step RT-PCR reaction is known to be one of the important factors that determine the detection sensitivity.

[0008] In the case of norovirus testing, it is usually necessary to simultaneously detect multiple target RNAs such as GI type norovirus (hereinafter, also referred to as norovirus G1 type, or simply GI, G1, etc.) and GII type norovirus (hereinafter, also referred to as norovirus G2 type, or simply GII, G2, etc.). In this case, it would be practically useful if the test could be performed by a one-step RT-PCR reaction that can reduce the risk of sample loss and scattering, the risk of contamination of other samples, and the like, in addition to being easy to operate. Norovirus is highly infectious, and it is said that infection is established with 10 to 100 viruses (Non-Patent Document 2). For this reason, a method capable of detecting norovirus with higher sensitivity is required, and in particular, in the one-step RT-PCR method, particularly in the multiplex one-step RT-PCR method targeting multiple target nucleic acids such as GI type and GII type, a method capable of detecting norovirus with higher sensitivity is desired. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] JP 2016-052265 A [Patent Document 2] JP 2015-119656 A [Patent Document 3] Patent No. 4437525 [Patent Document 4] Patent No. 4414648 [Patent Document 5] Patent No. 5354216 [Non-patent literature]

[0010] [Non-Patent Document 1] Ministry of Health, Labor and Welfare website, List of policies by field > Health and medicine > Food > Food poisoning > 4. Food poisoning statistics and survey results, (2) Past food poisoning occurrence status, Food poisoning occurrence status in 2018 (Heisei 30) (https: / / www.mhlw.go.jp / content / H30jokyo.xls) [Non-Patent Document 2] Ministry of Health, Labor and Welfare website, Current situation and countermeasures for food poisoning caused by norovirus (https: / / www.mhlw.go.jp / file / 06-Seisakujouhou-11130500-Shokuhinanzenbu / 0000105662.pdf) [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows representative examples of amplification curves under each primer condition in Test Example 1. Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention has been made in view of the above problems in the prior art. That is, an object of the present invention is to provide a means for detecting GII norovirus contained in a sample with higher sensitivity. [Means for solving the problem]

[0013] In light of the above circumstances, the inventor conducted intensive research and discovered that GII norovirus contained in a sample can be detected with higher sensitivity by using a specific oligonucleotide as a nucleic acid primer, thereby arriving at the present invention.

[0014] That is, the outline of the present invention is as follows. [Item 1] A method for detecting the presence of GII norovirus in a sample, comprising the steps of: (1) preparing a pair of nucleic acid primer sets consisting of the following combination of (I) and (II): (I) a nucleic acid primer consisting of a base sequence shown in any one of SEQ ID NOs: 6 to 37 or 42 to 54, or a base sequence complementary thereto; and (II) a nucleic acid primer consisting of a nucleotide sequence shown in SEQ ID NO: 4 or a nucleotide sequence complementary thereto; (2) preparing a mixture by mixing the sample with a one-step RT-PCR reaction solution containing the pair of nucleic acid primer sets and a thermostable DNA polymerase prepared in the step (1); and (3) After sealing the reaction vessel, a one-step RT-PCR reaction is carried out. [Item 2] The method according to Item 1, wherein the one-step RT-PCR reaction solution further contains a pair of nucleic acid primer sets for detecting GI norovirus, the pair comprising the following combination of (III) and (IV): (III) a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence complementary thereto; and (IV) A nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO:3 or a nucleotide sequence complementary thereto. [Item 3] The method according to Item 2, which is capable of detecting norovirus types GI and GII, respectively. [Item 4] The method according to any one of Items 1 to 3, wherein the one-step RT-PCR reaction solution further contains a probe comprising a nucleic acid having the base sequence shown in SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40, or a base sequence complementary thereto. [Item 5] The method according to item 4, wherein the probe is a TaqMan probe. [Item 6] The method according to any one of Items 1 to 5, wherein the sample has not been subjected to nucleic acid isolation or heat treatment. [Item 7] The method according to any one of Items 1 to 6, wherein steps (2) and (3) are carried out in the same vessel. [Item 8] The method according to any one of Items 1 to 7, characterized in that after sealing the reaction vessel in step (3), a one-step RT-PCR reaction is carried out without ever opening or closing the lid. [Item 9] A method according to any one of items 1 to 8, comprising carrying out a heat treatment in step (3) before and / or during the cycle reaction to disrupt the virus and expose nucleic acid within the virus and / or to activate a hot start enzyme in a nucleic acid amplification reaction. [Item 10] The method according to any one of items 1 to 9, wherein the sample is a blood sample, a stool sample, and / or a swab sample. [Item 11] The method according to any one of items 1 to 10, wherein the sample is a suspension in water, saline or a buffer solution. [Item 12] The method according to any one of Items 1 to 11, wherein the thermostable DNA polymerase is a thermostable DNA polymerase belonging to Family A. [Item 13] The method according to any one of Items 1 to 12, wherein the heat-stable DNA polymerase is at least one selected from the group consisting of Tth polymerase, HawkZ05 polymerase, and mutants thereof. [Item 14] The method according to any one of Items 1 to 13, wherein the one-step RT-PCR reaction solution further contains 1 mM or more of a divalent cation. [Item 15] The method according to any one of Items 1 to 14, wherein the one-step RT-PCR reaction is a one-enzyme-based one-step RT-PCR method. [Item 16] A nucleic acid primer for detecting GII type norovirus, comprising a base sequence shown in any one of SEQ ID NOs: 6 to 37 or 42 to 54, or a base sequence complementary thereto. [Item 17] The nucleic acid primer according to claim 16, A nucleic acid primer consisting of the base sequence shown in SEQ ID NO: 4 or a base sequence complementary thereto; A nucleic acid primer set for detecting GII type norovirus, comprising a combination of: [Item 18] A nucleic acid primer set for detecting GII type norovirus according to claim 17, a nucleic acid primer set for detecting GI norovirus, comprising a nucleic acid primer consisting of a nucleotide sequence shown in SEQ ID NO: 2 or a complementary nucleotide sequence thereto, and a nucleic acid primer consisting of a nucleotide sequence shown in SEQ ID NO: 3 or a complementary nucleotide sequence thereto; A nucleic acid primer set composition capable of detecting GI type norovirus and GII type norovirus, comprising: [Item 19] A thermostable DNA polymerase, A composition for testing a sample for GII type norovirus by a one-step RT-PCR reaction, comprising a pair of nucleic acid primer sets consisting of the following combinations of (I) and (II): (I) a nucleic acid primer consisting of a base sequence shown in any one of SEQ ID NOs: 6 to 37 or 42 to 54, or a base sequence complementary thereto; and (II) A nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO:4 or a nucleotide sequence complementary thereto. [Item 20] The composition according to Item 19, further comprising a pair of nucleic acid primer sets for detecting GI norovirus, the pair comprising the following combination of (III) and (IV): (III) a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence complementary thereto; and (IV) A nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO:3 or a nucleotide sequence complementary thereto. [Item 21] The composition described in Item 20, which is capable of detecting both GI and GII types of norovirus. [Item 22] A composition described in any one of Items 19 to 21, further comprising a probe comprising a nucleic acid having a base sequence shown in SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40, or a base sequence complementary thereto. [Item 23] The composition described in Item 22, wherein the probe is a TaqMan probe. [Item 24] The composition described in any one of Items 19 to 23, wherein the sample has not been subjected to nucleic acid isolation treatment or heat treatment. [Item 25] The composition of any of Items 19 to 24, wherein the sample is a blood sample, a stool sample, and / or a swab sample. [Item 26] The composition according to any one of Items 19 to 25, wherein the sample is a suspension in water, saline or a buffer solution. [Item 27] ​​The composition described in any one of Items 19 to 26, wherein the thermostable DNA polymerase is a thermostable DNA polymerase belonging to Family A. [Item 28] The composition described in any one of Items 19 to 27, characterized in that the heat-stable DNA polymerase is at least one selected from the group consisting of Tth polymerase, HawkZ05 polymerase, and mutants thereof. [Item 29] The composition according to any one of Items 19 to 28, further comprising 1 mM or more of a divalent cation. [Item 30] The composition described in any one of Items 19 to 29, wherein the one-step RT-PCR reaction is a one-enzyme-based one-step RT-PCR method. [Item 31] A kit for testing for GII norovirus in a sample, comprising the nucleic acid primer described in Item 16, the nucleic acid primer set described in Item 17, the nucleic acid primer set composition described in Item 18, and / or the composition described in any one of Items 19 to 30. Effect of the Invention

[0015] The present invention makes it possible to detect GII type norovirus with high sensitivity from a sample that may contain norovirus. Furthermore, when the nucleic acid primer for detecting GII type norovirus of the present invention is combined with a primer for detecting GI type norovirus to perform a multiplex RT-PCR reaction, it is unexpectedly possible to achieve an excellent effect that not only the detection sensitivity of GII type norovirus is improved, but also the detection sensitivity of GI type norovirus is improved. Furthermore, the present invention makes it possible to detect norovirus with high sensitivity from samples containing a large amount of impurities, such as fecal samples, blood samples, and environmental swabs. The present invention can also be used in life science research, clinical diagnosis, food hygiene testing, environmental testing, and the like. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention will be described in further detail below with reference to embodiments of the present invention, but the present invention is not limited thereto.

[0017] One embodiment of the present invention is a nucleic acid primer for detecting GII type norovirus contained in a sample, which is a nucleic acid primer consisting of a base sequence represented by any one of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54 (collectively referred to herein as "a base sequence represented by any one of SEQ ID NOs:6 to 37 or SEQ ID NOs:42 to 54") or a base sequence complementary thereto. The nucleic acid primer of the present invention may be a degenerate primer, and the mixed base pair designations are as follows according to the conventional art: R=A or G, M=A or C, W=A or T, S=C or G, Y=C or T, K=G or T, H=A or T or C, B=G or T or C, D=G or A or T, V=A or C or G, N=A or C or G or T.

[0018] The nucleic acid primer for detecting the GII type norovirus of the present invention corresponds to a part of the base sequence of the GII type norovirus RNA (corresponding to bases 4988 to 5048 of SEQ ID NO: 41). When a PCR reaction (including RT-PCR reaction, etc.) is performed using the nucleic acid primer of the present invention, the primer pair used in the PCR reaction may be a pair of two types of primers in which one primer is complementary to the DNA extension product of the other primer. In another embodiment, the above primers may be included in two or more pairs, that is, a so-called multiplex PCR. Furthermore, when the target nucleic acid is a subtype, a degenerate primer may be included. In a specific preferred embodiment of the present invention, a pair of nucleic acid primers can be used as a nucleic acid primer set in which the nucleic acid primer for detecting the GII type norovirus consisting of the base sequence shown in each of the above SEQ ID NOs is used as a forward primer and a conventionally known nucleic acid primer for detecting the GII type norovirus is used as a reverse primer.

[0019] Examples of conventionally known nucleic acid primers for detecting norovirus include, for example, the primers described in the notification (Food Safety Inspection No. 1105001) of the Inspection and Safety Division, Safety Department, Pharmaceuticals and Food Safety Bureau, Ministry of Health, Labor and Welfare (for example, the nucleic acid primer (COG2F) of SEQ ID NO: 1 and the nucleic acid primer (ALPF) of SEQ ID NO: 5 as forward primers for detecting GII type norovirus; the nucleic acid primer (COG2R) of SEQ ID NO: 4 as reverse primer for detecting GII type norovirus; the nucleic acid primer (COG1F) of SEQ ID NO: 2 as forward primer for detecting GI type norovirus; the nucleic acid primer (SEQ ID NO: 3) of SEQ ID NO: 3 as reverse primer for detecting GI type norovirus), but are not limited thereto. In a preferred embodiment, the nucleic acid primer of the present invention consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 6 to 37 or SEQ ID NOs: 42 to 54 or a nucleotide sequence complementary thereto, and the nucleic acid primer consisting of a nucleotide sequence shown in SEQ ID NO: 4 (COG2R) or a nucleotide sequence complementary thereto are used as a pair of nucleic acid primer sets, whereby GII type norovirus can be detected with higher sensitivity.

[0020] In a further embodiment, a multiplex RT-PCR reaction for detecting GI and / or GII noroviruses can be performed by combining a pair of nucleic acid primer sets consisting of a nucleic acid primer having a base sequence shown in SEQ ID NO: 2 (COG1F) or a base sequence complementary thereto with a pair of nucleic acid primer sets consisting of a base sequence shown in SEQ ID NO: 3 (COG1R) or a base sequence complementary thereto together with the pair of nucleic acid primer sets for detecting GII noroviruses of the present invention. By using the nucleic acid primers of the present invention, for example, GI noroviruses and / or GII noroviruses can be detected with good sensitivity in a multiplex one-step RT-PCR reaction.

[0021] A further embodiment of the present invention provides a nucleic acid primer set for detecting GII norovirus contained in a sample, the nucleic acid primer set comprising a combination of the following (I) and (II): (I) a nucleic acid primer consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 6 to 37 or 42 to 54, or a nucleotide sequence complementary thereto; and (II) A nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO:4 or a nucleotide sequence complementary thereto. In one embodiment, from the viewpoint of more reliably achieving the effects of the present invention, it is preferable to use the nucleic acid primer shown in (I) as a forward primer and the nucleic acid primer shown in (II) as a reverse primer. The nucleic acid primer set of the present invention can be suitably used, for example, in a one-step RT-PCR reaction.

[0022] Furthermore, the nucleic acid primer or nucleic acid primer set of the present invention is preferably used in combination with any pair of nucleic acid primer sets for detecting GI norovirus, and in particular, it is preferably used in combination with a pair of nucleic acid primer sets for detecting GI norovirus consisting of the following combinations (III) and (IV). (III) A nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence complementary thereto. (IV) A nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO:3 or a nucleotide sequence complementary thereto. When a pair of nucleic acid primer sets shown in (III) and (IV) above is used as a pair of nucleic acid primer sets for detecting GI norovirus, it is preferable, although not limited thereto, to use the nucleic acid primer shown in (III) above as a forward primer and the nucleic acid primer shown in (IV) above as a reverse primer, from the viewpoint of more reliably achieving the effects of the present invention.

[0023] The nucleic acid primer set of the present invention includes a pair of nucleic acid primer sets for detecting GI norovirus, and thus enables simultaneous detection of GI norovirus and GII norovirus with high sensitivity in a one-step RT-PCR reaction. Specifically, as shown in the results of the test examples described later, the use of the nucleic acid primers of the present invention for detecting GII norovirus has an unexpected and excellent effect of improving the detection sensitivity of GI norovirus when GI norovirus is also detected simultaneously.

[0024] When detecting norovirus using the nucleic acid primer of the present invention, the concentration of the nucleic acid primer of the present invention to be used is not particularly limited as long as the effect of the present invention is exhibited, but it is preferable that the concentration of the forward primer is 0.1 μM or more and 3 μM or less, and the concentration of the reverse primer is 0.1 μM or more and 3 μM or less, relative to the entire RT-PCR reaction solution. More preferably, the concentration of the forward primer is 0.1 μM or more and 2 μM or less, and the concentration of the reverse primer is 0.5 μM or more and 2 μM or less, thereby enabling more sensitive detection of GII type norovirus. The concentration of the other nucleic acid primer used in combination with the nucleic acid primer of the present invention to detect GII type norovirus, and the concentration of each nucleic acid primer to detect GI type norovirus when a pair of nucleic acid primer sets for detecting GI type norovirus are used are preferably used in the same amount as the nucleic acid primer concentration.

[0025] Norovirus is one of the viruses that cause food poisoning, and half of the food poisoning cases in Japan are caused by norovirus (Non-Patent Document 1). It is said that norovirus infection occurs through the feces or vomit of infected people, or through food.

[0026] It is known that noroviruses are largely classified into GI type noroviruses and GII type noroviruses according to their genotypes. It is desirable to distinguish between GI type noroviruses and GII type noroviruses from the viewpoint of collecting epidemiological data such as estimating the route of infection. The norovirus testing method using the nucleic acid primers of the present invention is highly useful in that it is possible to detect noroviruses with high sensitivity even when a pair of nucleic acid primer sets for detecting GI type noroviruses is combined, and thus it is possible not only to confirm the presence of GII type noroviruses but also to distinguish (distinguish) whether the infecting norovirus is GI type or GII type.

[0027] A method for testing for the presence of norovirus in a sample using the nucleic acid primer of the present invention is a method for testing for the presence of GII type norovirus, characterized by including at least the following steps: (1) preparing a pair of nucleic acid primer sets each consisting of a combination of nucleic acid primers each having a specific base sequence capable of detecting GII type norovirus; (2) preparing a mixture of a sample and the pair of nucleic acid primer sets prepared in the step (1) and a one-step RT-PCR reaction solution containing a thermostable DNA polymerase and, optionally, a reverse transcriptase; and (3) A step of performing a one-step RT-PCR reaction after sealing the reaction vessel. Here, the step of preparing a mixture in the step (2) can be performed, for example, by adding a one-step RT-PCR reaction solution containing a pair of nucleic acid primer sets including the nucleic acid primer of the present invention and a heat-stable DNA polymerase to a sample, but the order of mixing is not important.

[0028] As a further embodiment, the testing method of the present invention may further include, prior to step (1), a step (1') of exposing RNA from a norovirus capsid structure in a sample by subjecting a sample that may contain norovirus to a pretreatment such as a nucleic acid isolation and purification treatment or a chemical or thermodynamic treatment. The step (1') may be performed simultaneously during the one-step RT-PCR reaction in the previous step (3). It is preferable that the steps (1) to (3) or the steps (1') to (3) are all performed in the same container. That is, it is preferable that the whole or part of the mixed solution is not transferred to another container between the steps (1) to (3) or the steps (1') to (3). Furthermore, in step (3), it is preferable that the lid of the reaction container is not opened or closed after the reaction container is sealed. The sample used in step (1) may be a sample that may contain insoluble substances and has not been subjected to a prior centrifugation procedure or a suspension that has been previously suspended in water or a buffer solution, or a sample such as a stool sample may be directly added to the one-step RT-PCR reaction solution.

[0029] A further embodiment of the method for detecting norovirus using the nucleic acid primer or nucleic acid primer set of the present invention is a detection method using at least one type of labeled hybridization probe or double-stranded DNA-binding fluorescent compound. This allows the analysis of the amplified product to be monitored by monitoring a fluorescent signal instead of normal electrophoresis, reducing the analysis labor. Furthermore, there is no need to open the reaction vessel, and the risk of contamination can be reduced. It is also possible to identify virus subtypes by labeling each hybridization probe corresponding to the virus subtype with a different fluorescent dye. From the viewpoint of being able to show better specificity, it is preferable to use a hybridization probe, although this is not limited thereto.

[0030] Examples of double-stranded DNA-binding fluorescent compounds include SYBR (registered trademark) Green I, SYBR (registered trademark) Gold, SYTO-9, SYTP-13, SYTO-82 (Life Technologies), EvaGreen (registered trademark; Biotium), LCGreen (Idaho), and LightCycler (registered trademark) 480 ResoLight (Roche Applied Science).

[0031] Examples of the hybridization probe used in the present invention include TaqMan probes (also called Taq Man hydrolysis probes, etc.; U.S. Patent No. 5,210,015, U.S. Patent No. 5,538,848, U.S. Patent No. 5,487,972, and U.S. Patent No. 5,804,375), molecular beacons (U.S. Patent No. 5,118,801), and FRET hybridization probes (International Publication No. WO 97 / 46707, WO 97 / 46712, and WO 97 / 46714). Among these, Taq Man probes are preferably used. Examples of the base sequence of the probe for detecting norovirus include the sequences (SEQ ID NOs: 38 to 40) described in the notification (Food Safety Inspection No. 1105001) of the Surveillance and Safety Division, Safety Department, Pharmaceuticals and Food Safety Bureau, Ministry of Health, Labor and Welfare, and can be suitably used in the present invention, but are not limited thereto. In the above-described probe sequences, norovirus G1 type is detected by SEQ ID NO: 38 or 39, and norovirus G2 type is detected by SEQ ID NO: 40. Furthermore, when the target nucleic acid is a subtype, it may contain a degenerate sequence. The concentration of the fluorescently labeled probe is preferably 0.01 μM or more and 1.0 μM or less, more preferably 0.013 μM or more and 0.75 μM or less, and even more preferably 0.02 μM or more and 0.5 μM or less.

[0032] Examples of samples used in the present invention include, but are not limited to, feces (excreted feces, rectal feces), vomit, saliva, etc., and can be used for anything derived from a living body, and are particularly useful for detection from feces (excreted feces, rectal feces). The sample may be subjected to detection directly, or may be a sample obtained by suspending the sample in water, physiological saline, or a buffer solution in order to reduce the influence of impurities on the reaction and obtain more stable test results. Examples of the buffer solution include, but are not limited to, Hank's buffer solution, Tris buffer solution, phosphate buffer solution, glycine buffer solution, HEPES buffer solution, and Tricine buffer solution.

[0033] Another embodiment of the sample in the present invention is a sample derived from the environment, as represented by a swab test sample. The swab test is useful for clarifying the contamination route and grasping the contamination status of the facility environment. In the present invention, the swab test is not particularly limited, but for example, a sample obtained by wiping the relevant area or equipment with a cotton swab or the like, dissolving in water or a buffer solution, and concentrating with polyethylene glycol (PEG) precipitation. Specific examples of the swab test method include "Improvement of the norovirus test method for swab samples" (http: / / idsc.nih.go.jp / iasr / 32 / 382 / dj3824.html), but are not particularly limited and include a wide range of methods similar to this. Examples of the places to be swabbed include cooking utensils such as cutting boards, knives, dishcloths, and tableware, refrigerator handles, toilets, bathroom doorknobs, faucets such as washrooms, kitchens, toilets, and bathrooms, cookers' hands and fingers, bathrooms, toilets, washbasins, handrails, and rooms. In addition, although it is not a wipe test, the present invention can also be applied to concentrated sewage samples as an environmental test. These test samples contain a large amount of impurities such as dirt and dust from the test site, so the test method of the present invention, which can achieve high sensitivity, is useful for these tests.

[0034] In a particular preferred embodiment, the sample used in the present invention (e.g., a sample derived from a living organism (including secretions and excretions from a living organism), a sample derived from the environment, etc.) may be a sample that has not been subjected to a nucleic acid isolation procedure from the sample using a commercially available RNA purification kit or the like and / or a procedure of exposing RNA from a viral structure by prior heat treatment. In the method of the present invention, even if such a sample is one that does not undergo prior nucleic acid isolation or heat treatment, it can be subjected to a heat treatment before and / or during the cycle reaction of a one-step RT-PCR reaction to expose RNA from the viral structure and to the RT-PCR reaction. By using a sample that has not been subjected to prior nucleic acid isolation or heat treatment in this way, not only can the virus RNA be tested more easily and in a short time, but also the risk of contamination of other samples due to sample loss or carryover can be reduced. The effect is particularly remarkable in tests in which a large number of specimens are processed using feces as samples.

[0035] In a further specific embodiment, the sample used in the present invention may be a sample containing insoluble substances in its original state without prior centrifugation after suspending the sample in a liquid such as water, physiological saline or a buffer solution as necessary. If a sample containing insoluble substances without prior centrifugation is used in an RT-PCR reaction, as described above, the reaction solution will be highly turbid, and the insoluble substances contained in the reaction solution will scatter or absorb light, affect autofluorescence, etc., and there is a concern that the fluorescence intensity detectable during measurement with a real-time PCR machine will be significantly reduced, resulting in a decrease in sensitivity. However, since highly sensitive detection is possible by using the nucleic acid probe or nucleic acid probe set of the present invention, the present invention can obtain sufficient detection sensitivity even when using a sample containing such insoluble substances. For example, when the turbidity of the RT-PCR reaction solution after mixing with the sample is 0.01 to 5.0 Abs / μL at OD660, for example, when the turbidity is about 1.0 to 3.0 Abs / μL at OD660, it is expected that the present invention can detect GII type norovirus with good sensitivity.

[0036] The RT-PCR cycle in the step (3) consists of three steps: 1. heat treatment, 2. reverse transcription reaction, and 3. PCR. Before and after each step, a heat treatment step for activating a hot start enzyme may be included. The heat treatment step 1 includes a step of disrupting the virus to expose the nucleic acid in the virus, and / or a step of activating a hot start enzyme in a nucleic acid amplification reaction. By including these heat treatment steps, it is possible to expose (dissolve) RNA from the capsid structure of the virus. The temperature and time of the heat treatment step may be 60°C or higher and 1 second or longer, preferably 70°C, 30 seconds or longer, more preferably 80°C, 30 seconds or longer, and particularly preferably 85°C, 30 seconds or longer. The temperature of the reverse transcription reaction in 2 is determined by the reverse transcription activity of the heat-resistant DNA polymerase and the Tm values ​​of the primers and probes, and may be at least 25°C or higher. More preferably, it is 37°C or higher. The PCR of 3 may include three steps: [1] DNA denaturation by heat treatment (dissociation of double-stranded DNA into single-stranded DNA), [2] annealing of a primer to a template single-stranded DNA, and [3] extension of the primer using DNA polymerase. [2] and [3] may be performed at the same temperature to form a two-step process. In order to perform rapid RT-PCR, it is desirable to set a measurement program for the thermal cycler used in the RT-PCR reaction such that the total extension time of the steps [2] and [3] is 15 seconds or less, more preferably 10 seconds or less. In this specification, the term "PCR extension time" refers to the temperature set in the thermal cycler.

[0037] The one-step RT-PCR solution added in the step (2) is characterized by containing a heat-resistant DNA polymerase. The one-step RT-PCR reaction solution used in the step (2) may use a heat-resistant DNA polymerase that does not have reverse transcription activity. When a heat-resistant DNA polymerase that does not have reverse transcription activity is used, it can be used in combination with a reverse transcriptase to perform a two-enzyme one-step RT-PCR reaction. Furthermore, the one-step RT-PCR reaction solution used in the step (2) may use a heat-resistant DNA polymerase that has reverse transcription activity, and when such a heat-resistant DNA polymerase with reverse transcription activity is used, a one-enzyme one-step RT-PCR reaction can be performed. A DNA polymerase with reverse transcription activity is a DNA polymerase that has both the ability to convert RNA to cDNA and the ability to amplify DNA. Moreover, heat resistance means that the enzyme activity is not reduced by half or more even when heat treatment is performed at 70°C for 1 minute or more. The origin is not particularly limited, and examples thereof include Taq, Tth, Bst, Bca, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and mutants thereof. DNA polymerases having reverse transcription activity to date include DNA polymerase (Taq) derived from Thermus aquaticus, DNA polymerase (Tth) derived from Thermus thermophilus HB8, DNA polymerase (Z05) derived from Thermus sp Z05, DNA polymerase (Tma) derived from Thermotoga maritima, DNA polymerase (Bca) derived from Bacillus caldotenax, and DNA polymerase (Bst) derived from Bacillus stearothermophilus, and may be mutants that do not lose their reverse transcription activity and thermostable DNA polymerase activity.In addition, a mutant of Thermococcus kodakaraensis-derived DNA polymerase (KOD) is known to have reverse transcription activity (for example, RTX: reverse transcription xenopolymerase), and the heat-resistant DNA polymerase contained in the RT-PCR reaction solution added in the step (2) is not limited to such a heat-resistant DNA polymerase. Particularly preferred is a DNA polymerase belonging to family A, and preferably a heat-resistant DNA polymerase having reverse transcription activity selected from the group consisting of Taq, Tth, Z05, and mutants thereof. Of these, at least one selected from the group consisting of Tth polymerase, Hawk Z05 polymerase, and mutants thereof is preferred. The total amount of the heat-resistant DNA polymerase contained in the one-step RT-PCR reaction solution may be, for example, at least 4.2 ng / μL or more, preferably 5.0 ng / μL or more, and more preferably 5.8 ng / μL or more. Of these, preferably 8.3 ng / μL or more. The upper limit of the total amount of the heat-resistant DNA polymerase contained in the one-step RT-PCR reaction solution is not particularly limited, but as an example, it can be 20 ng / μL or less, and even if it is 16.7 ng / μL or less, the effect of the present invention can be sufficiently obtained. The amount of polymerase is a value quantified by the Bradford method or Nanodrop (Thermo Fisher), and may be roughly calculated from the safety data sheet (SDS). When a protein such as BSA is contained, it is preferable to calculate it by the latter method.

[0038] In the present specification, the term "mutant of thermostable DNA polymerase" refers to a mutant that has, for example, 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and especially preferably 99% or more sequence identity with respect to the amino acid sequence of the wild-type DNA polymerase from which it is derived, and has the activity of amplifying DNA like the wild-type DNA polymerase, and the activity of converting RNA to cDNA as necessary. Here, the method of calculating the identity of the amino acid sequence can be performed by any means known in the art. For example, it can be calculated using an analysis tool that is commercially available or available via an electric communication line (Internet). For example, the identity of the amino acid sequence can be calculated by using the default (initial setting) parameters in the homology algorithm BLAST (Basic local alignment search tool) of the National Center for Biotechnology Information (NCBI) http: / / www.ncbi.nlm.nih.gov / BLAST / . Furthermore, the mutant that can be used in the present invention may be a polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted and / or added (hereinafter, these are also collectively referred to as "mutation") in the amino acid sequence of the wild-type DNA polymerase from which it is derived, and may have the activity of converting RNA to cDNA and the activity of amplifying DNA, similar to the wild-type DNA polymerase. Here, "one or several" may be, for example, 1 to 80, preferably 1 to 40, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3, but is not particularly limited thereto.

[0039] In order to enhance the effect of suppressing non-specific reactions, the one-step RT-PCR reaction solution added in step (2) is preferably used in combination with an anti-DNA polymerase antibody or by introducing a heat-labile blocking group into the DNA polymerase by chemical modification, thereby suppressing the enzymatic activity of the DNA polymerase before carrying out the one-step RT-PCR reaction, and thus making it applicable to hot-start PCR.

[0040] The one-step RT-PCR reaction solution added in step (2) may contain, in addition to the heat-stable DNA polymerase, a buffer, an appropriate salt, a magnesium salt or a manganese salt, deoxynucleotide triphosphates, a primer pair corresponding to the detection target region of the viral RNA to be detected, and further, if necessary, additives.

[0041] The one-step RT-PCR solution added in the step (2) may contain a reverse transcriptase. In particular, when the one-step RT-PCR reaction solution in the step (1) uses a thermostable DNA polymerase that does not have reverse transcription activity, it is preferable that the one-step RT-PCR reaction solution further contains a reverse transcriptase. When the one-step RT-PCR reaction solution contains a reverse transcriptase different from the thermostable DNA polymerase, a two-enzyme one-step RT-PCR reaction can be performed. The reverse transcriptase contained in the one-step RT-PCR reaction solution is not particularly limited as long as it can convert RNA to DNA, and examples thereof include MMLV (Moloney Murine Leukemia Virus)-RT, AMV-RT (Avian Myeloblastosis Virus), HIV-RT, RAV2-RT, EIAV-RT, Carboxydothermus hydrogenoformam DNA polymerase) and mutants thereof. More preferred examples are MMLV-RT, AMV-RT, or mutants thereof. As with the thermostable DNA polymerase mutant, examples of reverse transcriptase mutants include mutants that are highly homologous to the amino acid sequence of the wild-type reverse transcriptase in terms of amino acid sequence identity, etc. (for example, mutants that show 90% or more amino acid sequence identity, or mutants that have one or several amino acid modifications in the wild-type amino acid sequence, etc.), but are not limited to these.

[0042] The buffer contained in the one-step RT-PCR solution added in the step (2) is not particularly limited, but includes Tris, Tricine, Bis-Tricine, Bicine, etc. The pH is adjusted to 6 to 9, more preferably 7 to 9, with sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, etc. The concentration of the buffer added is 10 to 200 mM, more preferably 20 to 150 mM. At this time, a salt solution is added to obtain ionic conditions suitable for the reaction. Examples of the salt solution include potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate, etc.

[0043] The dNTPs contained in the one-step RT-PCR solution added in the step (2) are dATP, dCTP, dGTP, and dTTP, each of which is added at 0.1 to 0.5 mM, most commonly at about 0.2 mM. Preventive measures against cross-contamination may be taken by using dUTP instead of and / or as a part of dTTP. When preventive measures against cross-contamination are taken, it is preferable to include Uracil-N-glycosylase (UNG).

[0044] Furthermore, the one-step RT-PCR solution added in the step (2) preferably contains a divalent cation. By containing a divalent cation in this way, more stable and high resistance to contamination can be obtained, and highly sensitive detection is possible. The divalent cation is not particularly limited, but examples thereof include magnesium ion, manganese ion, calcium ion, copper ion, iron ion, nickel ion, zinc ion, etc. Preferably, the divalent cation contains magnesium ion and manganese ion. In the present invention, when magnesium ion, manganese ion, etc. are added to the one-step RT-PCR reaction solution, magnesium or manganese may be added, or a salt thereof may be added. Examples of magnesium or a salt thereof include magnesium, magnesium chloride, magnesium sulfate, magnesium acetate, etc., and examples of manganese or a salt thereof include manganese, manganese chloride, manganese sulfate, manganese acetate, etc. Such magnesium, manganese, or a salt thereof is preferably added to the RT-PCR reaction solution at about 1 to 10 mM. From the viewpoint of easily obtaining a stable high sensitivity in the RNA virus detection method of the present invention, it is preferable to contain manganese or a salt thereof. In a specific embodiment, the RT-PCR reaction solution preferably contains 1 mM or more manganese or a salt thereof, more preferably 1.5 mM or more manganese or a salt thereof, and more preferably 2.0 mM or more manganese or a salt thereof.

[0045] The one-step RT-PCR reaction solution may further contain, as an additive, at least one selected from the group consisting of a quaternary ammonium salt having a structure in which three methyl groups are added to the amino group of an amino acid (hereinafter referred to as "betaine-like quaternary ammonium"), bovine serum albumin, sericin, BFP, glycerol, glycol, gelatin, and a surfactant.

[0046] The concentration of bovine serum albumin contained in the one-step RT-PCR reaction solution is not particularly limited, but is preferably at least 0.5 mg / ml or more, more preferably at least 1 mg / ml or more, based on the entire RT-PCR reaction solution. In a sample containing many impurities, a concentration of bovine serum albumin of preferably 2 mg / ml or more, more preferably 3 mg / ml or more, enables good detection. The upper limit is not particularly limited, but as an example, it can be 10 mg / ml or less.

[0047] The surfactant contained in the one-step RT-PCR reaction solution includes, but is not limited to, TritonX-100, TritonX-114, Tween20, Nonidet P40, Briji35, Briji58, SDS, CHAPS, CHAPSO, Emulgen420, etc. The concentration of the surfactant in the RT-PCR reaction solution is not particularly limited, but is preferably 0.0001% or more, more preferably 0.002% or more, and even more preferably 0.005% or more, to enable good detection. The upper limit is not particularly limited, but as an example, it can be 0.1% or less.

[0048] Examples of the betaine-like quaternary ammonium contained in the one-step RT-PCR reaction solution include, but are not limited to, betaine (trimethylglycine) and carnitine. The betaine structure is a compound having stable positive and negative charges in the molecule, and is considered to exhibit surfactant-like properties and cause destabilization of the virus structure. It is also known to promote nucleic acid amplification by DNA polymerase. The preferred concentration of the betaine-like quaternary ammonium is 0.1M to 2M, more preferably 0.2M to 1.2M.

[0049] Furthermore, it may be used in combination with substances known in the art to promote RT-PCR. Useful promoting substances include, but are not limited to, glycerol, polyols, protease inhibitors, single-stranded binding protein (SSB), T4 gene 32 protein, tRNA, sulfur- or acetate-containing compounds, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, ectoine, trehalose, dextran, polyvinylpyrrolidone (PVP), tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), polyethylene glycol, etc. In order to further reduce reaction inhibition, a chelating agent such as ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) may be included.

[0050] Another aspect of the present invention is a test kit or composition for GII type norovirus RNA, which includes the nucleic acid primer or nucleic acid primer set of the present invention. The kit may further include a nucleic acid primer or a nucleic acid primer set capable of detecting GI type norovirus RNA, thereby making it possible to prepare a test kit or composition capable of detecting GII type norovirus and GI type norovirus, respectively. In a particular preferred embodiment, the test kit or composition of the present invention may further include a heat-resistant DNA polymerase. Here, when the heat-resistant DNA polymerase does not have reverse transcription activity, it is preferable that the test kit or composition further includes a reverse transcriptase. In a further preferred embodiment, the norovirus test kit or composition of the present invention is a test kit or composition characterized in that it includes a reaction solution capable of performing one-step RT-PCR (one-step RT-PCR reaction solution).

[0051] The type and amount of the thermostable DNA polymerase or optionally reverse transcriptase, the type and amount of the primer or probe, the RNA virus to be tested, etc. used in this embodiment may be the same as those described in detail in the above-mentioned RNA testing method. The testing kit of the present invention may include, for example, an instruction manual explaining the method of use of the present invention. For example, the testing kit of the present invention may include the nucleic acid primer or nucleic acid primer set of the present invention for detecting GII type norovirus, the nucleic acid primer or nucleic acid primer set for detecting GI type norovirus, which may be included as necessary, the thermostable DNA polymerase, and the reverse transcriptase, all sealed in the same container or in separate containers, for example, in a single package, and may be provided in an embodiment including information on the method of use of the kit. EXAMPLES

[0052] The present invention will be described in detail below with reference to examples, although the present invention is not limited to the following examples.

[0053] Test Example 1. Study using each primer 1 (1) Preparation of norovirus solution The norovirus samples used were the Norovirus GI and GII Positive Controls (ZeptoMetrix, intact), which are norovirus specimens. G1 and G2 norovirus specimens were added at 250, 50, 25, and 10 copies per reaction. For the 10 copy condition, N=3 measurements were performed. (2) Reaction solution The reaction solution had the following basic composition, and norovirus was detected in the reaction solution using a one-enzyme, one-step RT-PCR. Primers were added at the concentrations shown in (3). Reaction solution 2x Master Mix (RNA-direct TM Realtime PCR Master mix (Toyobo accessory) 10x probe solution (TaqMan probe) (Norovirus Detection Kit G1 / G2 - High-Speed ​​Probe Detection - (Toyobo) Attachment) 20x Mn(OAc)2 (RNA-direct TM Realtime PCR Master mix (Toyobo accessory) The above-mentioned reagents and the primer solution described in (3) were mixed to prepare an RT-PCR reaction solution so that the final volume was 49 μL. 1 μL of the norovirus solution was added to the RT-PCR reaction solution, and RT-PCR was performed in a 50 μL reaction system. (3) Primer The following primers were added to the reaction solution so that the final concentrations during PCR were as follows. The primers listed in Table 1 were also added in the same manner. 0.5 μM COG1F (SEQ ID NO: 2) 0.5 μM COG1R (SEQ ID NO: 3) 0.5 μM COG2R (SEQ ID NO: 4) 0.1 μM ALPF (SEQ ID NO: 5) 0.5 μM [primers listed in Table 1] [Table 1]

[0054] (4) RT-PCR reaction conditions A real-time PCR reaction was carried out using BioRad's CFX96WELL DEEP with the following temperature cycle: Fluorescence reading was carried out at 60°C and 40 cycles of extension step. 90℃ 1 minute (heat treatment conditions) 58℃ 5 minutes (reverse transcription conditions) 95℃ 1 sec - 60℃ 10 sec 10 cycles (PCR) 95℃ 1 sec-60℃ 10 sec 40 cycles (PCR-fluorescence reading) (5) Results The results were evaluated in comparison with the standard condition 1 (conventional forward primer for detecting GII type norovirus; COG2F). The Ct values ​​obtained by the measurement are shown in Tables 2 and 3. Although it was impossible to detect 10 copies / reaction under condition 1, detection of 10 copies / reaction was confirmed under conditions other than condition 6 and condition 17. In addition, a representative example of an amplification curve is shown in FIG. 1. Even if the number of detected copies / reaction of 10 copies / reaction did not change as in condition 6, the amount of non-specific amplification was reduced, improving reactivity and improving the rise of the amplification curve. In addition, by using the nucleic acid primer of the present invention, it was unexpectedly shown that the detection sensitivity of not only GII type but also GI type tends to be improved. [Table 2] [Table 3]

[0055] Test Example 2. Sensitivity Comparison in Actual Samples 1 (1) Sample Feces from 90 specimens, including both norovirus negative and positive specimens, were prepared into a 10% fecal suspension, which was then centrifuged (12,000 rpm, 5 min) to obtain the supernatant. (2) Reaction solution The reaction solution had the basic composition shown below, and norovirus was detected in the reaction solution using a one-enzyme, one-step RT-PCR. Reaction solution 2x Master Mix (RNA-direct TM Realtime PCR Master mix (Toyobo accessory) 10x probe solution (TaqMan probe) (Norovirus Detection Kit G1 / G2 - High-Speed ​​Probe Detection - (Toyobo) Attachment) 20x Mn(OAc)2(RNA-direct TM Realtime PCR Master mix (Toyobo accessory) The above-mentioned reagents and the primer solution described in (3) were mixed to prepare an RT-PCR reaction solution with a final volume of 49 μL. 1 μL of the supernatant of the fecal suspension prepared in (1) was added to the RT-PCR reaction solution, and a 50 μL reaction system was subjected to RT-PCR. (3) Primer The following primers were added to the reaction solution so that the final concentrations during PCR were as follows. The primers listed in Table 4 were also added in the same manner. 0.5 μM COG1F (SEQ ID NO: 2) 0.5 μM COG1R (SEQ ID NO: 3) 0.5 μM COG2R (SEQ ID NO: 4) 0.1 μM ALPF (SEQ ID NO: 5) 0.5 μM [primers listed in Table 4] [Table 4]

[0056] (4) RT-PCR reaction conditions A real-time PCR reaction was carried out using BioRad's CFX96WELL DEEP with the following temperature cycle: Fluorescence reading was carried out at 60°C and 40 cycles of extension step. 90℃ 1 minute (heat treatment conditions) 58℃ 5 minutes (reverse transcription conditions) 95℃ 1 sec - 60℃ 10 sec 10 cycles (PCR) 95℃ 1 sec-60℃ 10 sec 40 cycles (PCR-fluorescence reading) (5) Results The number of negative and positive samples under each primer condition was compared. The results were compared with condition 1 (conventional forward primer for detecting GII type norovirus; COG2F). As shown in Table 5, the number of positive samples increased in both G1 and G2 under conditions 3, 4, and 5, confirming improved sensitivity. Under condition 2, the number of positive G1 samples decreased compared to condition 1, but the number of positive G2 samples increased compared to condition 1. [Table 5]

[0057] Test Example 3. Sensitivity Comparison in Actual Samples 2 (1) Sample Feces from 85 specimens, including both norovirus negative and positive specimens, were prepared into a 10% fecal suspension, which was then centrifuged (12,000 rpm, 5 min) to obtain the supernatant. (2) Reaction solution The reaction solution had the basic composition shown below, and norovirus was detected in the reaction solution using a two-enzyme one-step RT-PCR. 2x reaction solution (Norovirus detection kit G1 / G2-High-speed probe detection Quick step-(Toyobo) accessory) 10x probe solution (TaqMan probe) (Norovirus detection kit G1 / G2 - High-speed probe detection Quick step - (Toyobo) accessory) 10x Enzyme Solution (Norovirus Detection Kit G1 / G2 - High-Speed ​​Probe Detection Quick Step - (Toyobo) Attachment) The above-mentioned reagents and the primer solution described in (3) were mixed to prepare an RT-PCR reaction solution so that the final volume was 45 μL. 1 μL of fecal suspension supernatant was added to 3 μL of pretreatment solution included in the Norovirus Detection Kit G1 / G2-High-Speed ​​Probe Detection Quick Step- (Toyobo), and 45 μL of RT-PCR reaction solution was added on top of the mixture of pretreatment solution and fecal suspension supernatant, and the 49 μL reaction system was subjected to RT-PCR. (3) Primer The following primers were added to the reaction solution so that the final concentrations during PCR were as follows. The primers listed in Table 6 were also added in the same manner. 0.5 μM COG1F (SEQ ID NO: 2) 0.5 μM COG1R (SEQ ID NO: 3) 0.5 μM COG2R (SEQ ID NO: 4) 0.1 μM ALPF (SEQ ID NO: 5) 0.5 μM [primers listed in Table 6] [Table 6]

[0058] (4) RT-PCR reaction conditions A real-time PCR reaction was carried out using BioRad's CFX96WELL DEEP with the following temperature cycle: Fluorescence reading was carried out at 52°C and 40 cycles of extension step. 42℃ 4 minutes (reverse transcription conditions) 95℃ 1 minute 95℃ 1 sec - 52℃ 10 sec 10 cycles (PCR) 95℃ 1 sec-52℃ 10 sec 40 cycles (PCR-fluorescence reading) (5) Results The number of negative and positive samples under each primer condition was compared. The results were compared with condition 1 (conventional forward primer for detecting GII type norovirus; COG2F). As shown in Table 7, the number of positive samples increased in both G1 and G2 under conditions 2 and 3, confirming the improvement in sensitivity. [Table 7]

[0059] Test Example 4. Study using each primer 2 (1) Preparation of norovirus solution The norovirus samples used were the Norovirus GI and GII Positive Controls (ZeptoMetrix, intact), which are norovirus specimens. G1 and G2 norovirus specimens were added at 50, 25, and 10 copies per reaction. (2) Reaction solution The reaction solution had the following basic composition, and norovirus was detected in the reaction solution using a one-enzyme, one-step RT-PCR. Primers were added at the concentrations shown in (3). Reaction solution 2x Master Mix (RNA-directTM Realtime PCR Master mix (Toyobo) accessory) 10x probe solution (TaqMan probe) (Norovirus Detection Kit G1 / G2 - High-Speed ​​Probe Detection - (Toyobo) Attachment) 20x Mn(OAc)2 (included with RNA-directTM Realtime PCR Master mix (Toyobo)) The above-mentioned reagents and the primer solution described in (3) were mixed to prepare an RT-PCR reaction solution so that the final volume was 49 μL. 1 μL of the norovirus solution was added to the RT-PCR reaction solution, and RT-PCR was performed in a 50 μL reaction system. (3) Primer The following primers were added to the reaction solution so that the final concentrations during PCR were as follows: Additionally, the primers shown in Table 1 were added in the same manner. 0.5 μM COG1F (SEQ ID NO: 2) 0.5 μM COG1R (SEQ ID NO: 3) 0.5 μM COG2R (SEQ ID NO: 4) 0.1 μM ALPF (SEQ ID NO: 5) 0.5 μM [primers listed in Table 8] [Table 8]

[0060] (4) RT-PCR reaction conditions A real-time PCR reaction was carried out using BioRad's CFX96WELL DEEP with the following temperature cycle: Fluorescence reading was carried out at 60°C and 40 cycles of extension step. 90℃ 1 minute (heat treatment conditions) 58℃ 5 minutes (reverse transcription conditions) 95℃ 1 sec - 60℃ 10 sec 10 cycles (PCR) 95℃ 1 sec-60℃ 10 sec 40 cycles (PCR-fluorescence reading) (5) Results The results were evaluated in comparison with the standard condition 1 (conventional forward primer for detecting GII type norovirus; COG2F). The Ct values ​​obtained in the measurement are shown in Table 9. Although it was impossible to detect 10 copies / reaction under condition 1, detection of 10 copies / reaction was confirmed under all conditions from 2 to 14. Furthermore, unexpectedly, the use of the nucleic acid primer of the present invention showed a tendency for the detection sensitivity to improve not only for GII type but also for GI type. [Table 9]

[0061] Test Example 5. Sensitivity Comparison in Actual Samples 3 (1) Sample Feces from 24 specimens, including both norovirus negative and positive specimens, were prepared into a 10% fecal suspension, which was then centrifuged (12,000 rpm, 5 min) to obtain the supernatant. (2) Reaction solution The reaction solution had the basic composition shown below, and norovirus was detected in the reaction solution using a one-enzyme, one-step RT-PCR. Reaction solution 2x Master Mix (RNA-directTM Realtime PCR Master mix (Toyobo) accessory) 10x probe solution (TaqMan probe) (Norovirus Detection Kit G1 / G2 - High-Speed ​​Probe Detection - (Toyobo) Attachment) 20x Mn(OAc)2 (included with RNA-directTM Realtime PCR Master mix (Toyobo)) The above-mentioned reagents and the primer solution described in (3) were mixed to prepare an RT-PCR reaction solution with a final volume of 49 μL. 1 μL of the supernatant of the fecal suspension prepared in (1) was added to the RT-PCR reaction solution, and a 50 μL reaction system was subjected to RT-PCR. (3) Primer The following primers were added to the reaction solution so that the final concentrations during PCR were as follows: Additionally, the primers shown in Table 10 were added in the same manner. 0.5 μM COG1F (SEQ ID NO: 2) 0.5 μM COG1R (SEQ ID NO: 3) 0.5 μM COG2R (SEQ ID NO: 4) 0.1 μM ALPF (SEQ ID NO: 5) 0.5 μM [primers listed in Table 10] [Table 10]

[0062] (4) RT-PCR reaction conditions A real-time PCR reaction was carried out using BioRad's CFX96WELL DEEP with the following temperature cycle: Fluorescence reading was carried out at 60°C and 40 cycles of extension step. 90℃ 1 minute (heat treatment conditions) 58℃ 5 minutes (reverse transcription conditions) 95℃ 1 sec - 60℃ 10 sec 10 cycles (PCR) 95℃ 1 sec-60℃ 10 sec 40 cycles (PCR-fluorescence reading) (5) Results A comparison was made between the number of negative and positive samples under each primer condition. The results were compared with the conventional forward primer for detecting GII type norovirus (COG2F). As shown in Table 11, the number of positive samples for both G1 and G2 increased under all conditions 1 to 6, confirming improved sensitivity. (The total number of samples in the table exceeds 24 samples due to the inclusion of G1 and G2 coinfection samples.) [Table 11] [Industrial Applicability]

[0063] The present invention is suitable for use in molecular biology research, as well as in clinical testing and testing for food hygiene control.

Claims

1. A method for detecting the presence of GII norovirus in a sample, comprising the steps of: (1) preparing a pair of nucleic acid primer sets consisting of the following combination of (I) and (II): (I) a nucleic acid primer consisting of a base sequence represented by any one of SEQ ID NOs: 6 to 37 or 42 to 54; and (II) a nucleic acid primer consisting of the base sequence shown in SEQ ID NO: 4; (2) preparing a mixture by mixing the sample with a one-step RT-PCR reaction solution (excluding those containing an organic polar solvent) containing the pair of nucleic acid primer sets and a thermostable DNA polymerase prepared in the step (1); and (3) After sealing the reaction vessel, a one-step RT-PCR reaction is carried out: Here, the one-step RT-PCR reaction solution further comprises a pair of nucleic acid primer sets for detecting GI norovirus, the pair comprising the following combination of (III) and (IV): (III) a nucleic acid primer consisting of the base sequence shown in SEQ ID NO: 2; and (IV) A method comprising a nucleic acid primer consisting of the base sequence shown in SEQ ID NO:

3.

2. The method according to claim 1, which is capable of detecting norovirus types GI and GII, respectively.

3. The method according to claim 1 or 2, wherein the one-step RT-PCR reaction solution further contains a probe comprising a nucleic acid consisting of the base sequence represented by SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:

40.

4. The method of claim 3 , wherein the probe is a TaqMan probe.

5. The method according to any one of claims 1 to 4, wherein the sample has not been subjected to any nucleic acid isolation treatment or heat treatment.

6. The method according to any one of claims 1 to 5, wherein steps (2) and (3) are carried out in the same vessel.

7. The method according to any one of claims 1 to 6, wherein after the reaction vessel is sealed in the step (3), a one-step RT-PCR reaction is carried out without ever opening or closing the lid.

8. The method according to any one of claims 1 to 7, further comprising carrying out a heat treatment before and / or during the cycle reaction in order to disrupt the virus and expose nucleic acid within the virus and / or to activate a hot start enzyme in a nucleic acid amplification reaction.

9. The method according to any one of claims 1 to 8, wherein the sample is a blood sample, a stool sample and / or a swab sample.

10. 10. The method according to any one of claims 1 to 9, wherein the sample is a suspension in water, saline or a buffer solution.

11. The method according to any one of claims 1 to 10, wherein the thermostable DNA polymerase is a thermostable DNA polymerase belonging to family A.

12. 12. The method according to claim 1, wherein the thermostable DNA polymerase is at least one selected from the group consisting of Tth polymerase, Hawk Z05 polymerase, and mutants thereof.

13. The method according to any one of claims 1 to 12, wherein the one-step RT-PCR reaction solution further comprises 1 mM or more divalent cations.

14. The method according to any one of claims 1 to 13, wherein the one-step RT-PCR reaction is a one-enzyme-based one-step RT-PCR method.

15. a nucleic acid primer set for detecting GII type norovirus, comprising a nucleic acid primer having a base sequence shown in any one of SEQ ID NOs: 6 to 37 or 42 to 54 and a nucleic acid primer having a base sequence shown in SEQ ID NO: 4; a nucleic acid primer set for detecting GI norovirus, comprising a nucleic acid primer having a base sequence shown in SEQ ID NO: 2 and a nucleic acid primer having a base sequence shown in SEQ ID NO: 3; The nucleic acid primer set composition is capable of detecting GI type norovirus and GII type norovirus, and is for use in a one-step RT-PCR reaction using a one-step RT-PCR reaction solution that does not contain an organic polar solvent, and contains

16. The composition of claim 15, further comprising a thermostable DNA polymerase.

17. The composition according to claim 15 or 16, capable of detecting norovirus types GI and GII, respectively.

18. The composition according to any one of claims 15 to 17, further comprising a probe comprising a nucleic acid having a base sequence as shown in SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO:

40.

19. The composition of claim 18 , wherein the probe is a TaqMan probe.

20. The composition according to any one of claims 15 to 19, wherein the sample has not been subjected to any nucleic acid isolation treatment or heat treatment.

21. 21. The composition of any of claims 15 to 20, wherein the sample is a blood sample, a stool sample, and / or a swab sample.

22. 22. The composition of any of claims 15 to 21, wherein the sample is a suspension in water, saline or a buffer solution.

23. The composition according to any one of claims 15 to 22, wherein the thermostable DNA polymerase is a thermostable DNA polymerase belonging to family A.

24. 24. The composition according to any one of claims 15 to 23, wherein the thermostable DNA polymerase is at least one selected from the group consisting of Tth polymerase, Hawk Z05 polymerase, and mutants thereof.

25. 25. The composition of any one of claims 15 to 24, wherein the composition further comprises at least 1 mM of a divalent cation.

26. The composition according to any one of claims 15 to 25, wherein the one-step RT-PCR reaction is a one-enzyme-based one-step RT-PCR method.

27. A kit for testing a sample for GII type norovirus, comprising the composition according to any one of claims 15 to 26.

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