JP2016154468A - Method of detecting norovirus gii group - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and reagent for quickly and accurately detecting DNA of norovirus GII group.SOLUTION: Use of a pair of primer sets designed to be able to specifically amplify the ORF area of norovirus GII group enabling cDNA detection of the norovirus GII group.SELECTED DRAWING: None

Description

The present invention relates to an oligonucleotide for the rapid, reliable and simple detection of Norovirus GII group, a method for detecting Norovirus using the oligonucleotide, and a reagent for carrying out the method.

Norovirus is a virus having a single-stranded RNA of about 7500 bases in the genome. There are three Open Reading Frames (ORFs) on the genome, and it is known that ORF1 encodes nonstructural protein, ORF2 encodes structural protein 1 (VP1), and ORF3 encodes structural protein 2 (VP2). .

The genome base sequences of Norovirus are very diverse and are currently classified into five gene groups (GI to GV) based on their homology. Among these, it is said that there are three groups of GI, GII, and GIV that are pathogenic to humans, most of which are GI or GII groups. Recently, many GII / 4 have been detected.

Immunological methods and RT-PCR methods are used to detect norovirus (Patent Documents 1 to 4, Non-Patent Document 1). Immunological methods are often used mainly in medical tests, and RT-PCR is widely used in both medical and food tests.

The immunochromatography method can obtain a test result in about 15 minutes, so that a quick test is possible. However, its detection sensitivity is inferior compared with RT-PCR. For this reason, immunochromatography can be used to identify patients with norovirus infection, but it is difficult to detect those who have not developed virus.

The RT-PCR method is a method with excellent detection sensitivity. There are four steps: Norovirus RNA preparation, RNA reverse transcription, PCR of reverse transcripts, and detection of PCR products. A general RT-PCR method has a disadvantage that it takes two hours or more when all these steps are performed. For example, Patent Document 2 describes an example of norovirus detection using a norovirus-specific primer and a tackman probe, but in this example, more than 160 minutes are spent only in a PCR reaction for amplifying a reverse transcript. .

  In addition to the RT-PCR method, the detection method of the amplification product may be a problem in the inspection using the nucleic acid amplification method. In Patent Document 4, after performing RT-PCR with a Norovirus-specific primer, amplification products are detected by agarose gel electrophoresis. However, this method may cause contamination due to carryover of amplification products.

Japanese Patent No. 3887340 Japanese Patent No. 4414648 Japanese Patent No. 4437525 JP2011-78358

Journal of Clinical Microbiology, 2003, 41, p. 1548-1557

The problem to be solved by the present invention is to shorten the inspection time and prevent carry-over contamination in the RT-PCR method.

As a result of intensive studies in view of the above problems, the present inventors have found that norovirus GII type detection in a closed system using specific primers and probes is possible. Furthermore, it has been found that RT-PCR can be performed in a shorter time than the conventional method using the primer and probe, and the present invention has been completed. That is, the present invention provides the following.

[Section 1]
A method for detecting a nucleic acid of Norovirus GII group comprising detecting a nucleic acid amplification reaction and an amplification product obtained by the amplification reaction, comprising all the following steps (1) to (4), wherein step (2) A method for detecting a nucleic acid of Norovirus GII group, wherein (4) is completed within 1 hour, and step (2) and subsequent steps are performed without opening the reaction system.
(1) Step (2) to obtain DNA derived from Norovirus GII group by performing a reverse transcription reaction on Norovirus GII group RNA from a sample containing Norovirus Group II, In a reaction system comprising a nucleic acid primer group comprising at least one pair of nucleic acid primer sets, one type of nucleic acid probe, and a DNA polymerase having 3′-5 ′ exonuclease activity, PCR is performed so that the total reaction time is within 50 minutes. Step 3 to obtain amplification product (3) Form a complex of the nucleic acid amplification product obtained in step (2) and one or more nucleic acid probes capable of forming a complex with part or all of the nucleic acid amplification product Step (4) step of detecting the complex obtained in step (3)
[Section 2]
Item 2. The method according to Item 1, wherein at least one of the nucleic acid probes used in step (3) is a cytosine at least one of the nucleic acids, the cytosine is fluorescently labeled, and the labeled fluorescence Has a property of quenching when the nucleic acid probe is bound to a nucleic acid as a template, and emitting light when the nucleic acid probe is free without binding to other nucleic acid. A method for detecting nucleic acids of Norovirus GII group.
[Section 3]
Item 3. The method according to Item 1 or 2, wherein the step (4) is the following (4 ').
(4 ′) The presence or absence of dissociation between the nucleic acid amplification product forming the complex and the nucleic acid probe by performing a melting curve analysis by gradually increasing the temperature of the reaction system containing the complex obtained in step (3) Detecting process
[Section 4]
Item 4. The method according to any one of Items 1 to 3, further comprising the following features (i) and / or (ii):
(I) The nucleic acid primer set in step (2) is at least one pair selected from a combination of a forward primer selected from the following (A) and a reverse primer selected from the following (B).
(A) It has a base sequence that is 85% or more homologous to a base sequence having 112 'of the base sequence represented by SEQ ID NO: 1 as the 3' end and any base from the 81st to 90th of the sequence as the 5 'end Nucleic acid primer (B) A base sequence that is 85% or more homologous to a base sequence having the 3 'end at the 321st base sequence shown in SEQ ID NO: 2 and the 5' end at any of the 291 to 300th bases of the sequence Nucleic acid primer (ii) One type of nucleic acid probe used in step (3) is a nucleic acid probe corresponding to one of the following (C) or (D).
(C) 85% of the base sequence having the 153rd or 154th arbitrary base of the base sequence represented by SEQ ID NO: 1 as the 3 'end and the 135th to 139th arbitrary base of the sequence as the 5' end A nucleic acid probe having a homologous base sequence.
(D) The base sequence represented by SEQ ID NO: 2 has an arbitrary base of 350 to 352 as the 3 ′ end, and an arbitrary base of 330 to 340 of the sequence as the 5 ′ end and 85% of the base sequence A nucleic acid probe having a homologous base sequence.
[Section 5]
A nucleic acid probe having any of the following characteristics (C) or (D):
(C) 85% of the base sequence having the 153rd or 154th arbitrary base of the base sequence represented by SEQ ID NO: 1 as the 3 'end and the 135th to 139th arbitrary base of the sequence as the 5' end A nucleic acid probe having a homologous base sequence.
(D) The base sequence represented by SEQ ID NO: 2 has an arbitrary base of 350 to 352 as the 3 ′ end, and an arbitrary base of 330 to 340 of the sequence as the 5 ′ end and 85% of the base sequence A nucleic acid probe having a homologous base sequence.
[Section 6]
Item 6. The nucleic acid probe according to Item 5, wherein the nucleic acid probe has a base sequence represented by any of SEQ ID NOs: 9 to 16.
[Section 7]
A nucleic acid primer set comprising at least one pair of nucleic acid primers, wherein the pair is a combination of a forward primer selected from the following (A) and a reverse primer selected from the following (B): Nucleic acid primer set.
(A) It has a base sequence that is 85% or more homologous to a base sequence having 112 'of the base sequence represented by SEQ ID NO: 1 as the 3' end and any base from the 81st to 90th of the sequence as the 5 'end Nucleic acid primer (B) A base sequence that is 85% or more homologous to a base sequence having the 3 'end at the 321st base sequence shown in SEQ ID NO: 2 and the 5' end at any of the 291 to 300th bases of the sequence Nucleic acid primer [Item 8]
The forward primer is selected from the oligonucleotide having the base sequence represented by any of SEQ ID NOs: 3 to 5, and the reverse primer is selected from the oligonucleotide having the base sequence represented by any of SEQ ID NOs: 6 to 8, Item 8. The nucleic acid primer set according to Item 7.
[Claim 9]
A primer / probe set for detecting a Norovirus GII group, comprising the nucleic acid primer set according to Item 7 or Item 8 and the nucleic acid probe according to Item 5 or Item 6.
[Section 10]
Item 10. The method according to any one of Items 1 to 4, comprising the nucleic acid primer set according to Item 7 or Item 8, the nucleic acid probe according to Item 5 or Item 6, or the primer / probe set according to Item 9. Kit to carry out.

According to the present invention, it is possible to detect a Norovirus GII group reverse transcription product within one hour and without releasing the nucleic acid amplification product.

It is a figure which shows the result of Example 1. It is a figure which shows the result of Example 1. It is a figure which shows the result of the comparative example 1. It is a figure which shows the result of the comparative example 1. It is a figure which shows the result of the comparative example 1. It is a figure which shows the result of Example 2. It is a figure which shows the result of the comparative example 2. It is a figure which shows the result of the comparative example 2. It is a figure which shows the result of the comparative example 2.

  The present invention relates to a nucleic acid primer (primer) for nucleic acid amplification of a partial region of the ORF of Norovirus GII group, a nucleic acid probe (probe), and a method for performing detection and mutation discrimination of Norovirus GII group using these, and The present invention relates to a kit for carrying out the method.

  In the present invention, the partial region of ORF refers to NCBI accession No. Refers to the base sequence listed in Regions 4921 to 5400 of AF145896. The sequence is SEQ ID NO: 1. SEQ ID NO: 2 is a complementary sequence of SEQ ID NO: 1.

[Nucleic acid primer set]
In the detection method of Norovirus GII group of the present invention, the nucleic acid primer set for amplifying a region specific to Norovirus GII group comprises at least one pair of nucleic acid primers.
In this specification, “one pair of nucleic acid primer sets” is composed of one type (one sequence) of forward primers and one type (one sequence) of reverse primers.
When multiple pairs of nucleic acid primer sets are included, either the forward primer or the reverse primer may be common.

The nucleic acid primer set is not particularly limited as long as it is used for nucleic acid amplification of a partial region of the ORF of the Norovirus GII group. Preferably, the forward primer is composed of the following (A), and the reverse primer is the following: It is a nucleic acid primer set consisting of (B).
(A) It has a base sequence that is 85% or more homologous to a base sequence having 112 'of the base sequence represented by SEQ ID NO: 1 as the 3' end and any base from the 81st to 90th of the sequence as the 5 'end Nucleic acid primer (B) A base sequence that is 85% or more homologous to a base sequence having the 3 'end at the 321st base sequence shown in SEQ ID NO: 2 and the 5' end at any of the 291 to 300th bases of the sequence Nucleic acid primer

If the nucleotide sequence of the nucleic acid primer (A) is the 112th base of the nucleotide sequence represented by SEQ ID NO: 1 at the 3 'end, the 5' end is an arbitrary one of the 81st to 90th positions of SEQ ID NO: 1. It may be a base. Preferably, the 5 ′ end is the 83rd to 89th positions of SEQ ID NO: 1.
If the base sequence of the nucleic acid primer (B) is the 321st base of the base sequence represented by SEQ ID NO: 2 at the 3 'end, the 5' end is the 291st to 300th positions of SEQ ID NO: 2. Any base may be used. Preferably, the 5 ′ end is the positions 294 to 297 of SEQ ID NO: 2.

  In addition, the forward primer and the reverse primer constituting the nucleic acid primer set can be used for nucleic acid amplification by binding to the nucleic acid having the base sequence represented by SEQ ID NO: 1 or 2, and different bases from SEQ ID NO: 1 or 2 It may have an array. Moreover, a mixed base may be included in the bases constituting the nucleic acid primer. However, when the nucleotide sequence of the nucleic acid primer is greatly different from SEQ ID NO: 1 or 2, the specificity of the nucleic acid primer is lowered, and the possibility of erroneous amplification of nucleic acids other than Norovirus GII group DNA is increased. Therefore, the nucleic acid primer preferably has 85% or more identity with a partial base sequence of SEQ ID NO: 1 or SEQ ID NO: 2. More preferably 86%, more preferably 87%, more preferably 88%, more preferably 89%, more preferably 90%, more preferably 91%, more preferably 92%, more preferably 93%, more preferably Is 94%, more preferably 95%, more preferably 96%, more preferably 97%, more preferably 98%, more preferably 99%, more preferably 100%.

Various methods are known for calculating the identity of base sequences. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet).
In the present specification, nucleotide sequence homology is obtained by performing a search using blastn as a program and setting various parameters to default values in the homology algorithm Advanced BLAST 2.1 of the National Center for Biotechnology Information (NCBI). The value (%) is calculated.

As known methods, Patent Document 2 and Non-Patent Document 1 disclose a method for amplifying and detecting a similar region. The forward primer used in the method described in the above literature has the 3 ′ end at the 108th base of SEQ ID NO: 1 in this specification. The base sequence of the forward primer is shown as SEQ ID NO: 17 in this specification.
As a result of experiments, the inventors have found that the forward primer having the base sequence with the 3 ′ end at the 112th position is more forward than the forward primer described in the above document than the forward primer having the base sequence represented by SEQ ID NO: 17. Also found excellent detection sensitivity.

The reverse primer is also disclosed in Patent Document 2 and Non-Patent Document 1. The forward primer used in the method described in the above literature has the 5 ′ end at the 301st base of SEQ ID NO: 2 in the present specification. The base sequence of the reverse primer is shown as SEQ ID NO: 18 in this specification.
As a result of experiments, the inventors have described that a reverse primer having a base sequence in which the 5 ′ end is the 294th to 300th base is more described than the reverse primer having the base sequence represented by SEQ ID NO: 18. It was discovered that the detection sensitivity was better than the reverse primer.

  As a nucleic acid primer set excellent in such detection sensitivity, in particular, a base sequence represented by any one of SEQ ID NOs: 3 to 5 as a forward primer and a base sequence represented by any one of SEQ ID NOs: 6 to 8 as a reverse primer Combinations can be exemplified.

[Nucleic acid probe]
The nucleic acid probe used in the present invention is for detecting the ORF region of Norovirus GII group, and forms a complex by hybridizing with part or all of the nucleic acid amplification product amplified by the nucleic acid primer set. There is no particular limitation as long as it is possible. More preferably, it is a nucleic acid probe that specifically forms a complex only with the nucleic acid amplification product and does not form a complex with a nucleic acid having other base sequence, more preferably a part of the nucleic acid amplification product or A nucleic acid probe having the same or complementary base sequence as the entire base sequence.

Such a nucleic acid probe is preferably a nucleic acid probe having the characteristics (C) or (D).
(C) 85% of the base sequence having the 153rd or 154th arbitrary base of the base sequence represented by SEQ ID NO: 1 as the 3 'end and the 135th to 139th arbitrary base of the sequence as the 5' end A nucleic acid probe having a homologous base sequence.
(D) The base sequence represented by SEQ ID NO: 2 has an arbitrary base of 350 to 352 as the 3 ′ end, and an arbitrary base of 330 to 340 of the sequence as the 5 ′ end and 85% of the base sequence A nucleic acid probe having a homologous base sequence.

If the base sequence of the nucleic acid probe of (C) is the 153rd or 154th base of the base sequence represented by SEQ ID NO: 1 at the 3 'end, the 5' end is the 135th to 139th base of SEQ ID NO: 1. Any base in it may be used. More preferably, the 5 ′ end is the 136th to 138th positions of SEQ ID NO: 1.
In addition, the nucleotide sequence of the nucleic acid probe of (D) is that the 3 'end is any base in the 350th to 352rd base sequences represented by SEQ ID NO: 2, and the 5' end is SEQ ID NO: 2. Any base in the 330th to 340th positions may be used. More preferably, the 5 ′ end is the 332-338th position of SEQ ID NO: 2, and more preferably the 333-335th position.

  If the nucleic acid probes of (C) and (D) form a complex with the nucleic acid amplification product, a part of the nucleic acid probe base sequence is different from the base sequence represented by SEQ ID NO: 1 or 2. Also good. However, when the nucleotide sequence of the nucleic acid probe is greatly different from SEQ ID NO: 1 or 2, the specificity of the nucleic acid probe is lowered, and the possibility of forming a complex with a product other than the nucleic acid amplification product is increased. Therefore, the nucleic acid probe preferably has 85% or more identity with a partial base sequence of SEQ ID NO: 1 or SEQ ID NO: 2. More preferably 86%, more preferably 87%, more preferably 88%, more preferably 89%, more preferably 90%, more preferably 91%, more preferably 92%, more preferably 93%, more preferably Is 94%, more preferably 95%, more preferably 96%, more preferably 97%, more preferably 98%, more preferably 99%, more preferably 100%.

  An example of such a nucleic acid probe is a nucleic acid probe represented by any one of the nucleotide sequences of SEQ ID NOs: 9 to 16.

  The nucleic acid probe may or may not be labeled with a nucleic acid. In the case of labeling, the position and number of the nucleic acid to be labeled are not limited, but preferably the nucleic acid at the end of the nucleic acid probe is labeled, and more preferably, one of the ends is labeled. is there. More preferably, the base of the terminal nucleic acid to be labeled is cytosine.

  The labeling substance is not particularly limited, but is preferably a fluorescent substance, more preferably a fluorescent substance that exhibits fluorescence alone and quenches when it forms a hybrid with the target nucleic acid. Examples of the fluorescent substance include, but are not limited to, fluorescein, phosphor, rhodamine, polymethine dye derivatives, and the like. Examples of commercially available fluorescent dyes include BODIPY FL (trademark, manufactured by Molecular Probes), FluorePrime (trademark, Amersham). Pharmacia), Fluoredite (trademark, manufactured by Millipore), FAM (ABI), Cy3 and Cy5 (Amersham Pharmacia), TAMRA (Molecular Probes), carboxyrhodamine 6G (CR6G), and the like.

[Method for detecting norovirus GII group]
The method for detecting Norovirus GII group of the present invention is characterized by detecting DNA obtained by reverse transcription of Norovirus GII group RNA contained in a sample.

One aspect of the method includes a method characterized by including all of the following steps (1) to (4).
(1) Step (2) to obtain DNA derived from Norovirus GII group by performing a reverse transcription reaction on Norovirus GII group RNA from a sample containing Norovirus Group II, PCR is performed in a reaction system including a nucleic acid primer group including at least one pair of nucleic acid primer sets, one type of nucleic acid probe, and a DNA polymerase having 3′-5 ′ exonuclease activity so that the total reaction time is within 50 minutes. Step (3) of obtaining amplification product Step of forming nucleic acid amplification product obtained in step (2) and one type of nucleic acid probe complex capable of forming a complex with part or all of the nucleic acid amplification product ( 4) Step of detecting the complex obtained in step (3) The above-mentioned nucleic acid primer sets and nucleic acid probes used in the method are used Kill.

  The norovirus detection method of the present invention is not particularly limited except that the above steps are included. If norovirus DNA can be detected, a new step may be added to the above steps.

Step (1) can be performed using a commercially available reverse transcriptase. As the reverse transcriptase, a commercially available enzyme alone or a commercially available reverse transcriptase kit may be used. Examples of reverse transcriptase and reverse transcriptase kit include PrimeScript. Reverse Transcriptase (manufactured by Takara Bio), PrimeScript RT-PCR Kit (Takara Bio), (manufactured by Promega) M-MLV Reverse Transcriptase RNase H Minus, SuperScriptII Revrse Transcriptase (Life Ltd. Technologies), SuperScriptIII Revrse Transcriptase (Life Ltd. Technologies), RiverTra-Plus- (Toyobo), Rev rTra Ace -α- (Toyobo Co., Ltd.), ReverTra Ace qPCR RT Kit (Toyobo Co., Ltd.) and the like.
The reverse transcription reaction can be performed by the method described in the protocol attached to the reverse transcriptase or the reverse transcription reaction kit, or other conventionally known methods.

  As the nucleic acid primer used in the reverse transcription reaction of step (1), the nucleic acid primer described in (A) or (B) which is the nucleic acid primer of the present invention can be used, and in particular, the reverse primer of (B) is used. It is preferable. Moreover, a random primer generally used in reverse transcription reaction of RNA can be used. Although the base length in particular of a random primer is not restrict | limited, 4 bases or more and 12 bases or less are preferable. More preferably, they are 5 bases or more and 11 bases or less, More preferably, they are 6 bases or more and 10 bases or less.

  Although step (2) can be carried out with or without opening the reaction system, step (2) is preferably carried out without opening. “Open” means that the nucleic acid amplification product is exposed by, for example, opening a reaction container lid. “Do not open” means that the reaction container is not opened and the nucleic acid amplification product is not exposed to the outside.

As one aspect of the step (4) of the method, there is the following step (4 ′).
(4 ′) The presence or absence of dissociation between the nucleic acid amplification product forming the complex and the nucleic acid probe by performing a melting curve analysis by gradually increasing the temperature of the reaction system containing the complex obtained in step (2) Detecting process

  The time from the start of the steps (1), (2), (3), and (4) or the completion of the steps (1), (2), (3), and (4 ') is not particularly limited. It is preferable to finish (2) to (4) or (2) to (4 ′) within one hour. More preferably, it is within 55 minutes, is within 50 minutes, and is within 45 minutes. This time refers to the time from the start of PCR in step (2) to the completion of complex detection in step (4) or detection of the presence or absence of dissociation in (4 ′), and the reaction used in step (2) Liquid preparation time is not included.

  Of the steps (1), (2), (3), and (4), or (1), (2), (3), and (4 '), the time from the start of step (2) to completion is as follows. Preferably it is within 50 minutes. More preferably, it is 45 minutes, 40 minutes, 35 minutes, and 30 minutes. This time refers to the time from the start of the PCR in step (2) to the completion, and does not include the preparation time of the reaction solution used in step (2).

  Of the steps (1), (2), (3), and (4), or (1), (2), (3), and (4 '), the time from the start of step (1) to completion Although not particularly limited, it is preferably within 40 minutes, more preferably within 35 minutes, and within 30 minutes. This time refers to the time from the start of the reverse transcription reaction in step (1) to the completion thereof, and does not include the preparation time of the reaction solution used in step (1).

  As the nucleic acid primer set and the nucleic acid probe for amplifying and detecting the ORF of Norovirus GII group used in the method, those described above can be used. Furthermore, for example, for the purpose of amplifying and detecting a nucleic acid different from the DNA of Norovirus GII group, addition of a nucleic acid primer set or a nucleic acid probe different from the nucleic acid primer set or nucleic acid probe is not particularly limited.

[Amplification of test nucleic acid]
The test nucleic acid in the present invention may be, for example, a single strand or a double strand. In the case of a double strand, for example, it is preferable to include a step of dissociating the double strand into a single strand by heating in order to hybridize a test nucleic acid and a probe to form a hybrid.

  The type of the test nucleic acid is not particularly limited, and examples thereof include DNA, RNA such as total RNA and mRNA, and the like. Examples of the test nucleic acid include nucleic acids contained in a sample such as a biological sample. This sample may be used as it is, or may be diluted with an appropriate solution. Suitable solutions include water, saline, buffer solution, alkaline aqueous solution, acidic aqueous solution, nucleic acid extraction reagent, surfactant, organic solvent and the like.

The biological sample is not particularly limited, and examples thereof include feces, vomit, intestinal fluid, gastric fluid, rectal wiping solution, and pharyngeal wiping solution.
A method for collecting a sample, a method for preparing a nucleic acid such as DNA or RNA, and the like are not limited, and a conventionally known method can be employed.

  Subsequently, the isolated genomic DNA is used as a template to amplify a sequence containing a base site to be detected by a nucleic acid amplification method such as PCR using the above-described nucleic acid primer set. The specific nucleic acid amplification method is not particularly limited, and a known method can be used as appropriate. For example, PCR (Polymerase Chain Reaction) method, NASBA (Nucleic acid sequence based amplification) method, TMA (Transscription-mediated amplification (LMP) method, SDA (Strand Displacement Amplification method), SDA (Strand Displacement Amplification method). It is preferable to use the PCR method. In each of these methods, the conditions for the amplification reaction are not particularly limited, and can be performed by a conventionally known method.

  In the PCR method, usually, a three-stage reaction of a denaturation reaction, an annealing reaction, and an extension reaction is repeated. However, the two-stage reaction may be repeated by setting the same reaction conditions for the annealing reaction and the extension reaction. In the present invention, the reaction of the PCR method may be two steps, three steps, or four steps or more, preferably three steps.

The temperature and time conditions for each reaction are not limited, but the temperature for the denaturation reaction is preferably 90 to 100 ° C. The time for the denaturation reaction is preferably 0 to 10 seconds. Here, 0 seconds means that the temperature is set to a predetermined temperature for a moment and immediately proceeds to the next stage reaction, and does not mean that the denaturation reaction is not performed. The annealing reaction temperature is preferably 50 to 60 ° C., and the annealing reaction time is preferably 0 to 15 seconds. The temperature of the extension reaction is preferably 58 to 72 ° C., and the extension reaction time is preferably 1 to 15 seconds.

  Performing a denaturation reaction, an annealing reaction, and an extension reaction once each is called a PCR cycle, and the number of PCR cycles performed in the entire nucleic acid amplification reaction by the PCR method is called a cycle number. The number of cycles that can be carried out in the present invention is not particularly limited, but is preferably in the range of 35 to 100 times. The lower limit of the number of cycles is preferably 40 times, more preferably 50 times. The upper limit of the number of cycles is preferably 80 times, more preferably 70 times.

[DNA polymerase]
When the PCR method is used for nucleic acid amplification, the DNA polymerase to be used is not particularly limited, but α-type DNA polymerase is preferably used. The reason will be described below.

When a norovirus-derived DNA is amplified in a reaction system containing the probe of the present invention, the nucleic acid probe can bind to the sample norovirus-derived DNA or their amplification product during the nucleic acid amplification step. The nucleic acid probe bound to the Norovirus-derived DNA during the nucleic acid amplification step inhibits the nucleic acid amplification reaction by the nucleic acid primer and DNA polymerase.

PolI type DNA polymerases such as Taq DNA Polymerase are known to have 5'-3 'exonuclease activity. Due to this activity, when there is a nucleic acid bound to DNA derived from Norovirus as a template during the nucleic acid amplification reaction, the bound nucleic acid is degraded by exonuclease activity. For this reason, the nucleic acid probe in the reaction system may be reduced, causing a problem in the nucleic acid detection process. Therefore, it is not preferable to carry out the present invention using PolI type DNA polymerase.

On the other hand, α-type DNA polymerases such as KOD DNA Polymerase (derived from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1) do not have 5'-3 'exonuclease activity but have 3'-5' exonuclease activity. Therefore, the use of α-type DNA polymerase not only solves the above problem, but also exhibits high accuracy in the nucleic acid amplification step due to the 3′-5 ′ exonuclease activity.

Usually, α-type DNA polymerase has a 3 ′ → 5 ′ exonuclease activity, and therefore the nucleic acid amplification rate tends to be lower than that of PolI-type enzyme. However, although KOD DNA Polymerase is an α-type DNA polymerase, it has high DNA synthesis activity, has a DNA synthesis rate of 100 bases / second or more, and has excellent elongation efficiency. Therefore, it is preferable to use KOD DNA Polymerase (trade name, manufactured by Toyobo Co., Ltd.) among the α-type DNA polymerases for carrying out the present invention.

In addition, a mutant type in which α-type DNA polymerase is mutated to achieve a deoxyribonucleic acid synthesis rate of 100 bases / second or more, or a DNA polymerase composition in which the performance is achieved by a combination of wild type and / or mutant type Can be used as a DNA polymerase suitable for the practice of the present invention.
For example, as a DNA polymerase having a deoxyribonucleic acid synthesis rate of 100 bases / second or more in addition to the above KOD DNA Polymerase, “KOD FX (Toyobo, Trademark)”, “KOD-Plus- (Toyobo, Trademark)”, “KOD” Dash (trade name, manufactured by Toyobo Co., Ltd.), PrimeSTAR HS DNA polymerase (trade name, manufactured by Takara Bio Inc.) and the like can also be used.
Among these, KOD-Plus- having both high accuracy and DNA synthesis activity is desirable.

  By using the polymerase, PCR can be performed in a shorter time than conventional. In ordinary Taq polymerase, the deoxyribonucleic acid synthesis rate is said to be about 60 bases / second, but KOD DNA Polymerase and the like have a deoxyribonucleic acid synthesis rate exceeding 100 bases / second, so the extension time is reduced to half of the usual time. Can be shortened.

  In addition, PCR is further accelerated by using a nucleic acid amplification apparatus that performs a nucleic acid amplification reaction using a thin capillary reaction vessel, such as LightCycler (Roche Diagnostics) or GENECUBE (Toyobo). It is possible. By using these techniques, PCR within 1 hour is possible as shown in the examples of the present invention. However, even in this case, it is not possible to speed up the PCR and amplified nucleic acid detection reaction with all primers and probes as long as the enzymes and equipment are available. It is necessary to determine the right combination.

[Complex formation of nucleic acid amplification product and probe]
In the norovirus DNA detection method of the present invention, the nucleic acid amplification product obtained in step (1) is hybridized with a nucleic acid probe designed to form a complex with a part of the nucleic acid amplification product. Form.

The timing of adding a nucleic acid probe to a sample containing a nucleic acid amplification product is not particularly limited. For example, it may be added to the reaction system of the amplification reaction before, during or after the nucleic acid amplification reaction. May be.
Especially, since an amplification reaction and a detection reaction described later can be performed continuously, it is preferable to add them before the amplification reaction. Thus, when the probe is added before the nucleic acid amplification reaction, for example, as described later, it is preferable to add a fluorescent dye or a phosphate group to the 3 ′ end.

The probe may be added to a liquid sample containing a nucleic acid amplification product, or may be mixed with a nucleic acid amplification product in a solvent. The solvent is not particularly limited, and examples thereof include conventionally known solvents such as a buffer solution such as Tris-HCl, a solvent containing KCl, MgCl ₂ , MgSO ₄ , glycerol and the like, and a PCR reaction solution.

[Detection of complex between nucleic acid amplification product and probe]
In the step shown in (3) of the method, the method for detecting the obtained complex is not particularly limited. For example, a method by melting curve analysis as described in (3 ′) above can be mentioned.

In the case of melting curve analysis, for example, the following is performed.
When a solution containing double-stranded DNA is heated, the absorbance at 260 nm increases. This is because hydrogen bonds between both strands in double-stranded DNA are unwound by heating and dissociated into single-stranded DNA (DNA melting). When all the double-stranded DNAs are dissociated into single-stranded DNA, the absorbance is about 1.5 times the absorbance at the start of heating (absorbance of only double-stranded DNA), thereby melting. Can be determined to be completed.

  In the present invention, the measurement of the signal fluctuation accompanying the temperature change for performing the melting curve analysis can be performed by measuring the absorbance at 260 nm based on the principle as described above, but the signal of the label added to the probe of the present invention is measured. It is preferable to measure. For this reason, it is preferable to use a labeled probe as a probe used in the detection method of the norovirus GII group of the present invention.

  Examples of the labeled probe include a labeled probe that shows a signal alone and does not show a signal by hybridization, or a labeled probe that does not show a signal alone and shows a signal by hybridization. In the former probe, no signal is shown when a hybrid (double strand) is formed with the detection target sequence, and a signal is shown when the probe is released by heating. In the case of the latter probe, a signal is shown by forming a hybrid (double strand) with the sequence to be detected, and the signal decreases (disappears) when the probe is released by heating. Therefore, by detecting the signal from the label under signal-specific conditions (absorbance and the like), the progress of melting can be grasped in the same manner as the absorbance measurement at 260 nm.

  As a specific example of the labeled probe, for example, a probe that is labeled with a fluorescent dye, exhibits fluorescence alone, and fluorescence decreases (for example, quenches) by hybridization is preferable. Such a phenomenon is generally called a fluorescence quenching phenomenon. As a probe using this fluorescence quenching phenomenon, a probe generally called a guanine quenching probe is preferable. Such a probe is known as a so-called QProbe (registered trademark). A guanine quenching probe is, for example, a fluorescent dye designed so that the base at the 3 ′ end or 5 ′ end of an oligonucleotide becomes cytosine, and light emission becomes weaker when the base cytosine at the end approaches a complementary base guanine. It is a probe labeled at the end. In the probe of the present invention, for example, a fluorescent dye exhibiting a fluorescence quenching phenomenon may be bound to cytosine at the 3 ′ end of the oligonucleotide, or the 5 ′ end of the oligonucleotide is designed to be cytosine. It may be combined.

  In the probe used for the detection method of Norovirus GII group of the present invention, for example, a phosphate group may be added to the 3 'end. As will be described later, a test nucleic acid (target nucleic acid) for detecting the presence or absence of a gene can be prepared by a nucleic acid amplification method such as PCR. In this case, the probe of the present invention is allowed to coexist in the reaction system of the nucleic acid amplification reaction. be able to. In such a case, if a phosphate group is added to the 3 'end of the probe, the probe itself can be sufficiently prevented from extending due to the nucleic acid amplification reaction. The same effect can be obtained by adding a labeling substance as described above to the 3 'end.

  Dissociation of the obtained PCR amplification product and hybridization between the single-stranded DNA obtained by the dissociation and the labeled probe can be performed, for example, by changing the temperature of the reaction solution.

  The heating temperature in the dissociation step is not particularly limited as long as the amplification product can be dissociated, and is, for example, 85 to 98 ° C. The heating time is not particularly limited, but is usually 1 second to 10 minutes, preferably 1 second to 5 minutes.

  The dissociated single-stranded DNA and the labeled probe can be hybridized, for example, by lowering the heating temperature in the dissociation step after the dissociation step. As temperature conditions, it is 35-50 degreeC, for example.

  The volume and concentration of each composition in the reaction system (reaction system) of the hybridization process are not particularly limited. As a specific example, in the reaction system, the concentration of DNA is, for example, 0.01-100 μmol / L, preferably 0.1-10 μmol / L, and the concentration of the labeled probe is, for example, relative to the DNA A range satisfying the addition ratio is preferable, for example, 0.01 to 100 μmol / L, and preferably 0.01 to 10 μmol / L.

  Then, the temperature of the reaction solution is changed, and a signal value indicating the melting state of the hybrid formed of the amplification product and the labeled probe is measured. Specifically, for example, the reaction solution (hybridized body of the single-stranded DNA and the labeled probe) is heated, and a change in signal value accompanying a temperature rise is measured. As described above, when a probe labeled with a terminal C base (guanine quenching probe) is used, fluorescence is reduced (or quenched) in a hybridized state with single-stranded DNA, and in a dissociated state, Fluoresce. Therefore, for example, the hybrid formed body in which the fluorescence is decreased (or quenched) may be gradually heated, and the increase in the fluorescence intensity accompanying the temperature increase may be measured.

  The temperature range for measuring the fluctuation of the fluorescence intensity is not particularly limited. For example, the start temperature is room temperature to 85 ° C, preferably 25 to 70 ° C, and the end temperature is 40 to 105 ° C, for example. is there. Moreover, the rate of temperature rise is not particularly limited, but is, for example, 0.05 to 20 ° C./second, and preferably 0.08 to 5 ° C./second.

  The presence / absence of the test nucleic acid can be determined, for example, by measuring signal fluctuations during hybridization. That is, when a hybrid is formed by lowering the temperature of the reaction solution containing the probe, the signal fluctuation accompanying the temperature drop is measured.

  As a specific example, when a labeled probe (for example, a guanine quenching probe) that shows a signal alone and does not show a signal by hybridization, it emits fluorescence when the single-stranded DNA and the probe are dissociated. However, when a hybrid is formed due to a decrease in temperature, the fluorescence is reduced (or quenched). Therefore, for example, the temperature of the reaction solution may be gradually decreased to measure the decrease in fluorescence intensity accompanying the temperature decrease. On the other hand, when a labeled probe that does not show a signal alone and shows a signal by hybridization, it does not emit fluorescence when the single-stranded DNA and the probe are dissociated, but the hybrid is not released due to a decrease in temperature. Once formed, it will fluoresce. Therefore, for example, the temperature of the reaction solution may be gradually lowered and the increase in fluorescence intensity accompanying the temperature drop may be measured.

  In the present invention, in order to determine the genotype at the target base site, the signal variation may be analyzed and determined as a Tm (melting temperature) value.

[Primers, probes, detection kits]
The present invention also relates to a primer set, a probe, or a combination of the primer set and the probe used in the method for detecting the GII group of Norovirus described above.

  The present invention also relates to a norovirus GII group detection kit. The kit of the present invention includes the nucleic acid primer set and can be used for the norovirus GII group detection method. It is preferable that the kit further includes the nucleic acid probe and, in addition to that, a reagent necessary for a nucleic acid amplification reaction and / or a nucleic acid amplification product detection reaction as appropriate. Further, the kit is not limited to further containing reagents necessary for the reverse transcription reaction of RNA. Examples of reagents necessary for the reverse transcription reaction include reverse transcriptase and RNase inhibitor.

The method for measuring the activity of the DNA synthase used in the present specification is described below.
[DNA synthesis activity]
In the present invention, DNA synthesis activity refers to deoxyribonucleoside bound to deoxyribonucleic acid by covalently binding deoxyribonucleoside 5′-triphosphate α-phosphate to the 3′-hydroxyl group of the oligonucleotide or polynucleotide annealed to the template DNA. It refers to the activity of catalyzing the reaction of introducing 5′-monophosphate in a template-dependent manner.

When the enzyme activity is high, the activity is measured by diluting the sample with a storage buffer. In the present invention, 25 μl of the following A solution, 5 μl of each of B solution and C solution, and 10 μl of sterilized water are added to an Eppendorf tube and mixed with stirring. Then, 5 μl of the enzyme solution is added and reacted at 75 ° C. for 10 minutes. Thereafter, the mixture is ice-cooled, and 50 μl of E solution and 100 μl of D solution are added. This solution is filtered through a glass filter (Whatman GF / C filter), thoroughly washed with solution D and ethanol, and the radioactivity of the filter is measured with a liquid scintillation counter (manufactured by Packard) to incorporate nucleotides into the template DNA. taking measurement. One unit of enzyme activity is the amount of enzyme that incorporates 10 nmol nucleotides per 30 minutes into the acid-insoluble fraction under these conditions.
A: 40 mM Tris-HCl (pH 7.5)
16 mM magnesium chloride 15 mM dithiothreitol 100 μg / ml BSA
B: 2 μg / μl activated calf thymus DNA
C: 1.5 mM dNTP (250 cpm / pmol ^[3H] dTTP)
D: 20% trichloroacetic acid (2 mM sodium pyrophosphate)
E: 1 μg / μl carrier DNA

[3′-5 ′ exonuclease activity]
In the present invention, the 3′-5 ′ exonuclease activity refers to the activity of excising the 3 ′ terminal region of DNA and releasing 5′-mononucleotide.
The activity was measured using 50 μl of a reaction solution (120 mM Tris-HCl (pH 8.8 at 25 ° C.), 10 mM KCl, 6 mM ammonium sulfate, 1 mM MgCl ₂ , 0.1% Triton X-100, 0.001% BSA, 5 μg tritium-labeled E. coli DNA) is dispensed into a 1.5 ml Eppendorf tube and DNA polymerase is added. After reacting at 75 ° C. for 10 minutes, the reaction was stopped by cooling with ice, and then 50 μl of 0.1% BSA was added as a carrier, and then 100 μl of 10% trichloroacetic acid and 2% sodium pyrophosphate solution were added and mixed. To do. After leaving on ice for 15 minutes, the precipitate is separated by centrifugation at 12,000 rpm for 10 minutes. The radioactivity of 100 μl of the supernatant is measured with a liquid scintillation counter (manufactured by Packard), and the amount of nucleotide released in the acid-soluble fraction is measured.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.

[Example 1: Confirmation of usefulness of nucleic acid primer of the present invention]
(1) Preparation of sample A nucleic acid primer set obtained by mixing 5 pmol each of the nucleic acid primer of SEQ ID NO: 6 and the nucleic acid primer of SEQ ID NO: 7 from RNA of Norovirus known to be a GII group, and ReverseTra Ace qPCR RT Kit ( CDNA was synthesized by reverse transcription reaction at 37 ° C. for 15 minutes using 5 × RT Buffer and RT Enzyme Mix manufactured by Toyobo Co., Ltd. After the reaction, the reverse transcriptase was inactivated by heating at 98 ° C. for 5 minutes. The synthesized cDNA was adjusted to a concentration of about 100 copies / μl using 10 mM Tris buffer and used as a sample.
(2) Nucleic acid amplification and melting curve analysis The following reagents were added to the above samples, respectively, and nucleic acid amplification and melting curve analysis were performed under the following conditions to detect a complex of a nucleic acid probe and a nucleic acid amplification product. For analysis, GENECUBE (registered trademark) manufactured by Toyobo Co., Ltd. was used.

[Reagent for nucleic acid amplification]
A solution containing the following reagents was prepared. The combinations of nucleic acid primer sets are as listed in Table 1. (In Table 1, the numbers in the primer row and the probe row indicate the sequence numbers.)
100 μM forward primer (any one of SEQ ID NOs: 3 to 5) 0.25 μl
0.5 μl of 10 μM reverse primer (any one of SEQ ID NOs: 6 to 8)
0.3 μl of 10 μM nucleic acid probe (SEQ ID NO: 13, 3 ′ end labeled with BODIPY-FL)
KOD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 3 μl
PPD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 3 μl
Purified water 1.95 μl
Sample 1μl

[Nucleic acid amplification and melting curve analysis]
94 ° C, 30 seconds (more than one cycle)
97 ° C · 1 second 55 ° C · 5 seconds 63 ° C · 5 seconds (over 50 cycles)
94 ° C, 30 seconds, 39 ° C, 30 seconds, 39 ° C to 75 ° C (temperature rises at 0.09 ° C / second)

[result]
FIG. Nucleic acid amplification was performed using the nucleic acid primer set represented by 1 and 2 and the nucleic acid probe having the base sequence consisting of SEQ ID NO: 13, and the change in fluorescence intensity as the temperature increased thereafter was plotted on the horizontal axis of the graph. It is a figure showing an analysis result by making temperature and the vertical axis | shaft the differential value of a fluorescence signal. The nucleic acid probe used in this example is a quenching probe, which binds to and quenches the amplified target nucleic acid by placing it at 39 ° C. together with the nucleic acid amplification product. Thereafter, by gradually raising the temperature, the nucleic acid probe is released from the target nucleic acid and emits fluorescence. Therefore, if the amount of change in fluorescence changes positively in the process of gradually raising the temperature, this indicates that the nucleic acid probe bound to the target nucleic acid has been released.
In FIG. 1, peaks are observed from both combinations. Therefore, no. The nucleic acid primer of the combination of 1 and 2 can nucleic acid amplify cDNA reverse transcribed from Norovirus RNA, and the nucleic acid probe having the base sequence represented by SEQ ID NO: 13 can detect the cDNA It was shown that.
Furthermore, the time required from the start of the nucleic acid amplification reaction to the detection of the target nucleic acid by melting curve analysis was about 45 minutes.

FIG. 2 shows the combination of the nucleic acid primers shown in Table 1 It is the result of having performed nucleic acid amplification and melting curve analysis by changing to 3-5, respectively. In any case, the peak of the fluorescence differential value can be confirmed, and it was shown that cDNA derived from Norovirus of GII group was amplified and detected. Therefore, the nucleic acid primer of the present invention was reverse transcribed from RNA of Norovirus GII group. It was shown that the nucleic acid probe of the present invention can detect the nucleic acid amplification product. Further, the nucleic acid primer set used in the present invention is not limited to one type, and it was shown that a wide variety of nucleic acid primer combinations are possible. Moreover, it was shown that steps (2) to (4) of the detection method of the present invention can be completed within one hour.

[Comparative Example 1: Rapid verification of the method of the present invention]
Here, the method of the present invention was compared with the method of the prior document (Patent Document 2), and it was confirmed that the present invention can be carried out in a shorter time than the method of the prior document. The present invention and the method of the prior art are different in the DNA polymerase and the nucleic acid probe used. The present invention uses KOD DNA Polymerase and QProbe, and the method of the prior art uses Taq Polymerase and TaqMan Probe (registered trademark). Therefore, in this comparative example, the experiment was performed with four combinations shown in Table 2. As Taq Polymerase, rTaq Polymerase (manufactured by Toyobo) was used.

(1) Preparation of sample From Norovirus RNA known to be GII group, 5xRT Buffer, RT Enzyme Mix, Primer Mix (mixture of random primer and oligo (dT) primer) of ReverseTra Ace qPCR RT Kit (Toyobo) Was used for reverse transcription at 37 ° C. for 15 minutes to synthesize cDNA. After the reaction, the reverse transcriptase was inactivated by heating at 98 ° C. for 5 minutes. The synthesized cDNA was adjusted to a concentration of about 1000 copies / μl using 10 mM Tris buffer and used as a sample.

(2) Nucleic acid amplification Nucleic acid amplification was performed by the following two methods.
(2-1) Normal PCR / melting curve analysis Normal PCR and melting curve analysis were performed under the following conditions. Rotor-Gene Q (QIAGEN) was used for the analysis.
95 ° C for 3 minutes (1 cycle or more)
95 ° C · 10 seconds 55 ° C · 30 seconds (Green and Yellow fluorescence acquired)
72 ° C, 30 seconds (more than 55 cycles)
94 ° C, 30 seconds Cooling to 40 ° C 40 ° C to 80 ° C (Increase by 1 ° C, hold for 5 seconds every 1 ° C increase, acquire Green fluorescence)

(2-2) High-speed PCR and melting curve analysis High-speed PCR and melting curve analysis were performed under the following conditions. For the analysis, LightCycler 2.0 (Roche) was used.
95 ° C for 1 minute (more than one cycle)
97 ° C · 2 seconds 55 ° C · 5 seconds 68 ° C · 5 seconds (over 60 cycles)
94 ° C./60 seconds 40 ° C./60 seconds 40 ° C. to 80 ° C.

[Reagent for nucleic acid amplification]
A solution containing the following reagents was prepared. The nucleic acid primers SEQ ID Nos. 17 and 18 used here are nucleic acid primers described in Patent Document 2 and Non-patent Document. Combination No. SEQ ID NO: 19, which is a TaqMan Probe used in 1 and 2, is a nucleic acid probe described in Patent Document 2. The TaqMan Probe used in this comparative example was one in which the 5 ′ end was labeled with TET and the 3 ′ end was labeled with TAMRA.
For each reagent, 1-fold amount described below, that is, 8 μl, was used for LightCycler, and 2.5-fold amount, that is, 20 μl, described below was used for Rotor-Gene Q. In Rotor-Gene Q, 3 μl of purified water was further added. And as a sample cDNA, 2 μl of LightCycler and Rotor-Gene Q, that is, about 2000 copies equivalent was added.

(Reagent for combination No. 1)
0.5 μl of 10 μM forward primer (SEQ ID NO: 17)
0.5 μl of 10 μM reverse primer (SEQ ID NO: 18)
10 μM TaqMan Probe (SEQ ID NO: 19, 5 ′ end labeled with TET, 3 capture end labeled with TAMRA) 0.5 μl
rTaq Polymerase (Toyobo) 0.75 μl
Anti-rTaq Polymerase antibody (Toyobo) 0.75 μl
25 mM magnesium chloride 0.6 μl
10 × rTaq Buffer (manufactured by Toyobo) 1.0 μl
2 mM dNTP (Toyobo) 1.0 μl
Purified water 2.4 μl
8μl total

(Reagent for combination No. 2)
100 μM forward primer (SEQ ID NO: 17) 0.5 μl
0.5 μl of 10 μM reverse primer (SEQ ID NO: 18)
10 μM QProbe (SEQ ID NO: 13, 3 ′ end labeled with BODIPY-FL) 0.25 μl
rTaq Polymerase (Toyobo) 0.75 μl
Anti-rTaq Polymerase antibody (Toyobo) 0.75 μl
25 mM magnesium chloride 0.6 μl
10 × rTaq Buffer (manufactured by Toyobo) 1.0 μl
2 mM dNTP (Toyobo) 1.0 μl
2.65 μl of purified water
8μl total

(Reagent for combination No. 3)
0.5 μl of 10 μM forward primer (SEQ ID NO: 17)
0.5 μl of 10 μM reverse primer (SEQ ID NO: 18)
10 μM TaqMan Probe (SEQ ID NO: 19, 5 ′ end labeled with TET, 3 capture end labeled with TAMRA) 0.5 μl
KOD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 3 μl
25 μM magnesium sulfate (manufactured by Toyobo) 1.2 μl
Purified water 2.3 μl
8μl total

(Reagent for combination No. 4)
100 μM forward primer (SEQ ID NO: 17) 0.5 μl
0.5 μl of 10 μM reverse primer (SEQ ID NO: 18)
10 μM QProbe (SEQ ID NO: 13, 3 ′ end labeled with BODIPY-FL) 0.25 μl
KOD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 3 μl
25 μM magnesium sulfate (manufactured by Toyobo) 1.2 μl
Purified water 2.55 μl
8μl total

[result]
FIG. 3 shows the results of high-speed PCR / melting curve analysis performed by LightCycler (Roche). From FIG. 3, the enzyme / probe combination No. The clearest peak was obtained by the combination of 4, ie, KOD DNA Polymerase having 3′-5 ′ exonuclease activity and QProbe. Since the peak in the melting curve analysis indicates that a complex of the nucleic acid amplification reaction product and the nucleic acid probe exists, this peak is a combination No. 4 shows that an amplification product using Norovirus GII type cDNA as a template was obtained in the reaction system No. 4. On the other hand, the combination No. No peak was obtained for No. 2, and no. 1 and 3 have peaks but are weak, so in these reaction systems, amplification ability or detection ability is combination No. It is weaker than 4 and can be said to be unsuitable for high-speed PCR. From the start of PCR until the analysis result is obtained, it is completed within one hour. 4 is suitable as a method for measuring Norovirus GII group in a short time.

FIG. 4 shows the results of normal PCR / melting curve analysis using Rotor-Gene Q (Qiagen). Here, the enzyme / probe combination No. 3, that is, in the reaction system combining KOD DNA Polymerase and TaqMan Probe, a peak indicating detection of a cDNA amplification product is obtained although it is unclear. On the other hand, no. No. 1 shows a change in fluorescence value, but there is no clear peak. In the reaction system using 2 and 4, that is, QProbe, no peak was observed. In the experiment shown in FIG. 4, the reason why the combination No. 4 could not successfully detect the norovirus-derived DNA is because of the condition of the nucleic acid extension reaction (72 ° C., 30 seconds). The optimal extension reaction temperature of Taq Polymerase is 72 ° C., and the normal PCR conditions in this experiment are also matched. Since the Rotor-Gene Q measurement in this experiment was performed by imitating the example of Patent Document 2, the PCR conditions were also similar to those of the document. On the other hand, since the optimum temperature of KOD Polymerase is around 68 ° C., the Rotor-Gene Q measurement in this experiment is considered to have resulted in a decrease in amplification efficiency and an unclear detection result.
FIG. 5 is a trace of fluorescence during annealing with Rotor-Gene Q. Here, no. 1 and No. In the reaction system using 2, ie, TaqMan Probe, fluorescence was observed. The rise of the curve is No. 2 occurred in an overwhelmingly early stage. However, when purified water is used as a sample, no. No. 2 showed the same tendency, so no. The fluorescence in 2 is considered to be observed regardless of the amplification product. This is probably due to the fact that TaqMan Probe was decomposed by the 3′-5 ′ exonuclease activity of KOD DNA Polymerase and the fluorescent material emitted light regardless of the binding to the template DNA. Combination No. In 1, fluorescence was observed only when cDNA was used as a sample, but the rise of the curve was after about 40 cycles. It took more than 1 hour to confirm the rise of the curve. Even if the measurement under normal PCR conditions is performed in 1, it is impossible to measure the Norovirus GII group within 1 hour.
From the above, it was shown that the method of the present invention can detect the norovirus GII group more rapidly than the methods of the prior art.

[Example 2: Usefulness of nucleic acid probe of the present invention]
(1) Sample preparation (2) Nucleic acid amplification and melting curve analysis Both were performed in the same manner as in Example 1.

[Reagent for nucleic acid amplification]
A solution containing the following reagents was prepared.
100 μM forward primer (SEQ ID NO: 4) 0.5 μl
100 μM reverse primer (SEQ ID NO: 6) 0.1 μl
0.3 μl of 10 μM nucleic acid probe (SEQ ID NO: 9 or 14, 3 ′ end labeled with BODIPY-FL)
KOD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 3 μl
PPD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 3 μl
Purified water 2.1μl
Sample 1μl

[Nucleic acid amplification and melting curve analysis]
The same operation as in Example 1 was performed.

[result]
FIG. 6 shows nucleic acid amplification using a nucleic acid primer / probe combination in which the forward primer is SEQ ID NO: 4, the reverse primer is SEQ ID NO: 6, and the nucleic acid probe is SEQ ID NO: 9 or 14, and then the temperature rises FIG. 6 is a diagram showing the analysis result of the change in fluorescence intensity with the horizontal axis of the graph as the temperature and the vertical axis as the differential value of the fluorescence signal. As is apparent from FIG. 6, norovirus GII group RNA-derived cDNA could be detected using any nucleic acid probe, as in Example 1. From the above, it was shown that the nucleic acid probe that can be used in the present invention can have a plurality of base sequences instead of a single base sequence.

[Comparative Example 2: Performance comparison with nucleic acid primer described in prior literature]
(1) Preparation of sample From Norovirus RNA known to be GII group, 5xRT Buffer, RT Enzyme Mix, Primer Mix (mixture of random primer and oligo (dT) primer) of ReverseTra Ace qPCR RT Kit (Toyobo) Was used for reverse transcription at 37 ° C. for 15 minutes to synthesize cDNA. After the reaction, the reverse transcriptase was inactivated by heating at 98 ° C. for 5 minutes. The synthesized cDNA was adjusted to a concentration of about 100 copies / μl or 30 copies / μl using 10 mM Tris buffer, and used as a sample.
(2) Nucleic acid amplification and melting curve analysis Same as Example 1.

[reagent]
Solutions (1) and (2) containing the following reagents were prepared. The combinations of nucleic acid primers and nucleic acid probes are shown in Table 3. (In Table 3, the numbers in the primer column and the probe column indicate the sequence numbers.)

Solution (1)
100 μM nucleic acid primer (either one of SEQ ID NO: 3 or 17) 0.15 μl
0.25 μl of 10 μM nucleic acid primer (SEQ ID NO: 18)
0.3 μl of 10 μM nucleic acid probe (SEQ ID NO: 13, 3 ′ end labeled with BODIPY-FL)
KOD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 3 μl
PPD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 3 μl
cDNA (100 copies / μl) 1 μl
Purified water 2.3 μl

Solution (2)
100 μM nucleic acid primer (either one of SEQ ID NO: 4 or 5) 0.5 μl
100 μM nucleic acid primer (either one of SEQ ID NO: 6 or 18) 0.1 μl
0.3 μl of 10 μM nucleic acid probe (SEQ ID NO: 13, 3 ′ end labeled with BODIPY-FL)
KOD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 3 μl
PPD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 3 μl
cDNA (30 copies / μl) 1 μl
Purified water 2.1μl

[Nucleic acid amplification and melting curve analysis]
The same operation as in Example 1 was performed.

[result]
FIG. Using the nucleic acid primer / probe combination shown in 6 and 7 in the composition of the solution (1), nucleic acid amplification was performed, and the change in fluorescence intensity as the temperature increased thereafter was plotted with the horizontal axis of the graph being temperature and the vertical axis being It is a figure showing an analysis result as a differential value of a fluorescence signal. The forward primer of SEQ ID NO: 17 and the reverse primer of SEQ ID NO: 18 used here are nucleic acid primers described in Patent Document 2 and Non-patent Document.
From FIG. 7, it was shown that norovirus GII group-derived cDNA was not amplified in the nucleic acid primer set consisting of the combination of SEQ ID NOs: 17 and 18. On the other hand, it was shown that cDNA is amplified when the forward primer is changed to SEQ ID NO: 3, which is the nucleic acid primer described in the present invention.
8 to 9 show the nucleic acid primers in combination Nos. Described in Table 3. 8 is a result of performing nucleic acid amplification and melting curve analysis with the composition of solution (2), respectively changing to 8-11. Combination No. In No. 8, no detection peak was observed. Since the peak at 9 was also low, it was suggested that the nucleic acid primer sets constituting these combinations were inferior in amplification efficiency. On the other hand, combination no. 10 and no. No. 11 showed a detection peak, so the nucleic acid primer sets constituting these combinations were No. 11 8 and no. It was suggested that the amplification efficiency was superior to that of the 9 nucleic acid primer sets. No. 8 and no. No. 9 uses SEQ ID NO: 18 as a reverse primer. 10 and no. 11, since SEQ ID NO: 6 was used for the reverse primer, the difference in these results was considered to be due to the reverse primer, suggesting that the reverse primer of the present invention has better performance.

By using the present invention for genetic testing of the norovirus GII group, it is possible to detect the norovirus GII group within 1 hour and to easily determine the mutation at the same time.

Claims (10)

A method for detecting a nucleic acid of Norovirus GII group comprising detecting a nucleic acid amplification reaction and an amplification product obtained by the amplification reaction, comprising all the following steps (1) to (4), wherein step (2) A method for detecting a nucleic acid of Norovirus GII group, wherein (4) is completed within 1 hour, and step (2) and subsequent steps are performed without opening the reaction system.
(1) Step (2) to obtain DNA derived from Norovirus GII group by performing a reverse transcription reaction on Norovirus GII group RNA from a sample containing Norovirus Group II, In a reaction system comprising a nucleic acid primer group comprising at least one pair of nucleic acid primer sets, one type of nucleic acid probe, and a DNA polymerase having 3′-5 ′ exonuclease activity, PCR is performed so that the total reaction time is within 50 minutes. Step 3 to obtain amplification product (3) Form a complex of the nucleic acid amplification product obtained in step (2) and one or more nucleic acid probes capable of forming a complex with part or all of the nucleic acid amplification product Step (4) step of detecting the complex obtained in step (3) 2. The method according to claim 1, wherein one kind of nucleic acid probe used in step (3) is such that at least one of the terminal nucleic acids is cytosine, and the cytosine is fluorescently labeled. Fluorescence has the property of quenching when the nucleic acid probe is bound to a template nucleic acid and emitting light when the nucleic acid probe is free without binding to other nucleic acids. A method of detecting a nucleic acid of Norovirus GII group using a probe. The method according to claim 1 or 2, wherein the step (4) is the following (4 ').
(4 ′) The presence or absence of dissociation between the nucleic acid amplification product forming the complex and the nucleic acid probe by performing a melting curve analysis by gradually increasing the temperature of the reaction system containing the complex obtained in step (3) Detecting process The method according to any one of claims 1 to 3, further comprising the following features (i) and / or (ii): detecting a nucleic acid of Norovirus GII group.
(I) The nucleic acid primer set in step (2) is at least one pair selected from a combination of a forward primer selected from the following (A) and a reverse primer selected from the following (B).
(A) It has a base sequence that is 85% or more homologous to a base sequence having 112 'of the base sequence represented by SEQ ID NO: 1 as the 3' end and any base from the 81st to 90th of the sequence as the 5 'end Nucleic acid primer (B) A base sequence that is 85% or more homologous to a base sequence having the 3 'end at the 321st base sequence shown in SEQ ID NO: 2 and the 5' end at any of the 291 to 300th bases of the sequence Nucleic acid primer (ii) One type of nucleic acid probe used in step (3) is a nucleic acid probe corresponding to one of the following (C) or (D).
(C) 85% of the base sequence having the 153rd or 154th arbitrary base of the base sequence represented by SEQ ID NO: 1 as the 3 'end and the 135th to 139th arbitrary base of the sequence as the 5' end A nucleic acid probe having a homologous base sequence.
(D) The base sequence represented by SEQ ID NO: 2 has an arbitrary base of 350 to 352 as the 3 ′ end, and an arbitrary base of 330 to 340 of the sequence as the 5 ′ end and 85% of the base sequence A nucleic acid probe having a homologous base sequence. A nucleic acid probe having any of the following characteristics (C) or (D):
(C) 85% of the base sequence having the 153rd or 154th arbitrary base of the base sequence represented by SEQ ID NO: 1 as the 3 'end and the 135th to 139th arbitrary base of the sequence as the 5' end A nucleic acid probe having a homologous base sequence.
(D) The base sequence represented by SEQ ID NO: 2 has an arbitrary base of 350 to 352 as the 3 ′ end, and an arbitrary base of 330 to 340 of the sequence as the 5 ′ end and 85% of the base sequence A nucleic acid probe having a homologous base sequence. The nucleic acid probe according to claim 5, wherein the nucleic acid probe has a base sequence represented by any of SEQ ID NOs: 9 to 16. A nucleic acid primer set comprising at least one pair of nucleic acid primers, wherein the pair is a combination of a forward primer selected from the following (A) and a reverse primer selected from the following (B): Nucleic acid primer set.
(A) It has a base sequence that is 85% or more homologous to a base sequence having 112 'of the base sequence represented by SEQ ID NO: 1 as the 3' end and any base from the 81st to 90th of the sequence as the 5 'end Nucleic acid primer (B) A base sequence that is 85% or more homologous to a base sequence having the 3 'end at the 321st base sequence shown in SEQ ID NO: 2 and the 5' end at any of the 291 to 300th bases of the sequence Nucleic acid primer The forward primer is selected from the oligonucleotide having the base sequence represented by any of SEQ ID NOs: 3 to 5, and the reverse primer is selected from the oligonucleotide having the base sequence represented by any of SEQ ID NOs: 6 to 8, The nucleic acid primer set according to claim 7. A set of primers and probes for detecting Norovirus GII group, wherein the nucleic acid primer set according to claim 7 or 8 and the nucleic acid probe according to claim 5 or 6 are combined. The nucleic acid primer set according to claim 7 or 8, the nucleic acid probe according to claim 5 or 6, or the primer / probe set according to claim 9 according to any one of claims 1 to 4. Kit for carrying out the method.