JP2018078806A - Methods for detecting noroviral rna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods for detecting and distinguishing noroviral RNA genogroup I (GI) and genogroup II (GII) in a single closed vessel and at a constant temperature.SOLUTION: The invention provides a method for detecting and distinguishing noroviral RNA genogroup I (GI) and genogroup II (GII) in a sample comprising the steps of: (1) amplifying a specific base sequence that allows discrimination of noroviral RNA genogroup I (GI) and genogroup II (GII); and (2) detecting the amplified sequence, where the steps of amplification and detection are carried out in a single closed vessel and at a constant temperature.SELECTED DRAWING: None

Description

  The present invention quickly, sensitively and specifically sealed Norovirus RNA Genogroup I (hereinafter sometimes referred to as “GI”) and Genogroup II (hereinafter sometimes referred to as “GII”). The present invention relates to a single container and a method of detecting separately at a constant temperature.

  Norovirus is a virus belonging to the human caliciviridae family and has a single-stranded RNA of about 7000 bases in the genome. Norovirus is also referred to as Small Round Structured Virus (SRSV). Noroviruses are roughly classified into two gene groups according to genotype, namely genogroup I RNA (hereinafter referred to as “Norovirus GI RNA”) and genogroup II RNA (hereinafter referred to as “Norovirus GII RNA”). Further, for each gene group, norovirus GI RNA is currently distinguished into genotypes 1 to 9 and norovirus GII RNA is distinguished into genotypes 1 to 22.

About 20% of food poisoning reported in Japan is estimated to be caused by viruses. Norovirus is detected in about 80% or more of these cases of viral food poisoning. The main source of infection is food, often with raw oysters. In addition, norovirus is also detected in infants' (sporadic) acute gastroenteritis, suggesting the possibility of transmission from human to human. In order to stop the Norovirus epidemic, it is necessary to respond as quickly as possible. Based on the above, rapid testing for norovirus has become a major issue in public health and food quality control, and it is possible to detect all genotypes with high sensitivity and speed using gene amplification methods. Development of high inspection methods is desired. Furthermore, a test method for identifying and detecting Norovirus GI RNA and Norovirus GII RNA, which leads to identification of the infection route and the prevalent genes, is very useful for public health.
On the other hand, for detection of norovirus, a reagent for specific antibody detection ELISA using virus-like hollow particles of human calicivirus has been developed (Patent Document 1). However, the detection sensitivity is by no means high sensitivity.

  As a means for measuring norovirus with high sensitivity, there is a method of amplifying norovirus RNA by RT-PCR and measuring the amount of amplification product (Patent Document 2). In this method, generally, reverse transcription (RT) is used. A two-stage process is required: a process and a PCR process. This not only makes the operation complicated and deteriorates the reproducibility, but also increases the risk of secondary contamination. When the RT process and the PCR process are combined, it usually takes 2 hours or more, which is unsuitable for processing a large number of specimens and reducing examination costs.

  As a method for quantifying a target RNA, a Real-time RT-PCR method in which a PCR step carried out following an RT step is carried out in the presence of an intercalating fluorescent dye to measure an increase in fluorescence is widely used (non-patented). Document 1) has a problem that the method also detects non-specific amplification products such as primer dimers.

  Further, as a method for quantifying a target RNA that does not detect a non-specific amplification product, a real-time RT-PCR method using a hybridization probe such as a TaqMan probe is used as a PCR step performed subsequent to the RT step (Non-patent Document 2). However, as described above, when the RT process and the PCR process are combined, it usually takes 2 hours or more and is not quick. Furthermore, PCR has to raise and lower the reaction temperature abruptly, which has been a barrier for labor saving and cost reduction of the reaction apparatus at the time of automation.

  On the other hand, as a method for amplifying only RNA at a constant temperature, the NASBA method (see Patent Documents 3 and 4), the TMA method (see Patent Document 5), and the like have been reported. The RNA amplification method synthesizes double-stranded DNA containing a promoter sequence with a primer containing a promoter sequence, reverse transcriptase and, if necessary, ribonuclease H (RNase H) for target RNA, and RNA polymerase. RNA comprising a specific base sequence of target RNA is synthesized, and the RNA is subsequently subjected to a chain reaction that serves as a template for synthesizing double-stranded DNA containing a promoter sequence. Then, after RNA amplification, the amplified RNA is detected by electrophoresis or a hybridization method using a nucleic acid probe bound with a detectable label.

  As described above, the RNA amplification method is suitable for simple RNA measurement because it amplifies only RNA at a constant temperature and in one step. However, detection by a hybridization method or the like requires complicated operation and quantifies with high reproducibility. There is a problem that it cannot be done.

  As a method for simply amplifying and measuring RNA, the method of Ishiguro et al. The method is a nucleic acid probe labeled with an intercalating fluorescent dye, and when a complementary double strand is formed with a target nucleic acid, the intercalating fluorescent dye portion is intercalated into the complementary double strand portion. The RNA amplification method is carried out in the presence of a nucleic acid probe designed to change the fluorescence characteristics, and the change in the fluorescence characteristics is measured. Measurements can be performed simultaneously.

Each of the nucleic acid amplification methods is a method of amplifying a target RNA by a combination of oligonucleotides composed of a sense primer (first primer) and an antisense primer (second primer), and the combination is the nucleic acid amplification. It is well known that it has important implications for the efficiency and specificity of. However, since norovirus has extremely diverse genotypes, construction of primer sets that amplify all genotypes uniformly and efficiently, and oligonucleotides that distinguish and detect norovirus GI RNA and norovirus GII RNA, respectively. It is difficult to construct a probe set. In particular, when using an amplification method capable of performing amplification and detection of a target RNA under a relatively low temperature (preferably 40 to 50 ° C.), the primer / probe tends to form a higher-order structure. Therefore, construction of a primer / probe set is extremely difficult.
As described above, there are 9 genotypes in Norovirus GI RNA and 22 genotypes in Norovirus GII RNA, and the region where the base sequence is highly conserved is not sufficiently long. Therefore, it was difficult to detect Norovirus RNA rapidly and with high sensitivity regardless of the gene group / genotype by combining oligonucleotides each using one kind of the first primer and the second primer.

  Therefore, the inventors previously measured quickly and highly sensitively for a wide range of genotypes of Norovirus GI RNA by using a combination of oligonucleotides each using two or more types of the first primer and the second primer. (Patent Document 7). Moreover, it discovered that it can detect rapidly and highly sensitively with respect to the wide genotype of Norovirus GII RNA by using 2 or more types of oligonucleotide for a cutting | disconnection (patent document 8).

  Furthermore, by optimizing and combining oligonucleotides that can be detected quickly and with high sensitivity against a wide range of genotypes of Norovirus GI RNA and Norovirus GII RNA, Norovirus RNA can be used once regardless of gene group / genotype. A method for detecting the detection quickly and with high sensitivity has been found (Patent Document 9).

WO2000 / 079280 Japanese Patent No. 3752102 Japanese Patent No. 2650159 Japanese Patent No. 3152927 Japanese Patent No. 3241717 JP 2000-14400 A JP 2009-63 A JP 2009-17824 A JP 2010-4393 A

Kageyama T. et al. , Journal of Clinical Microbiology, 41, 1548-1557 (2003). Food Safety Supervision No. 1105001, November 5, 2003 Ishiguro T. et al, Anal. Biochem. , 314, 77-86 (2003)

  However, the above method cannot distinguish and detect Norovirus GI RNA and Norovirus GII RNA, and a method for distinguishing and detecting the gene group is required for public health.

  As a result of intensive studies on the above problems, the present inventors have found a method for distinguishing and detecting Norovirus GI RNA and Norovirus GII RNA quickly and with high sensitivity.

That is, the present invention is as follows.
[1] A method for distinguishing and detecting genogroup I and genogroup II of norovirus RNA present in a sample,
(1) a step of amplifying a specific base sequence capable of distinguishing between genogroup I and genogroup II in norovirus RNA, and (2) a step of detecting the amplified sequence,
Have
The method according to claim 1, wherein the amplifying step and the detecting step are performed in a single sealed container and at a constant temperature.
[2] The method according to [1], wherein the detecting step is performed by real-time monitoring.
[3] (1) A first primer and a second primer having a specific base sequence in Norovirus RNA as a template and having a promoter sequence added to either 5 ′ end, RNA-dependent DNA polymerase, ribonuclease H (RNase H And a double-stranded DNA comprising a promoter sequence and the specific base sequence downstream of the promoter sequence by a DNA-dependent DNA polymerase,
(2) A step of generating an RNA transcript having the specific base sequence or its complementary sequence by RNA polymerase using the double-stranded DNA as a template,
(3) a step of amplifying the RNA transcript in a chain manner by using the RNA transcript as a template for the subsequent double-stranded DNA synthesis;
(4) measuring the amount of the RNA transcript,
And the step (4) of measuring the amount of RNA transcript in the presence of a nucleic acid probe designed to change the signal property when a complementary double strand is formed with the target RNA. The nucleic acid probe is made by measuring a change, and the nucleic acid probe is an oligonucleotide probe consisting of 15 or more consecutive bases in the sequence shown in SEQ ID NO: 1 or its complementary sequence, and a continuous in the sequence shown in SEQ ID NO: 2. The method according to [1] or [2], which is an oligonucleotide probe comprising 15 bases or more or a complementary sequence thereof.

  Hereinafter, the present invention will be described in detail.

  In the present invention, the specific base sequence that can distinguish between GI and GII is a base sequence that is specifically found in each of Norovirus GI RNA and Norovirus GII RNA, and a base that exists in a region in which higher order structures are difficult to take. Is an array.

  Examples of the nucleic acid amplification method that can be employed in the amplification step include the LAMP method, TRC method, NASBA method, and TMA method, but the specific nucleotide sequence that exists in a region where the oligonucleotide probe used in the present invention does not easily take a higher order structure. Therefore, the TRC method, NASBA method, and TMA method, which can be carried out easily and quickly at a constant temperature (relatively low temperature), are preferred.

A particularly preferred amplification step uses a first primer having a sequence homologous to a part of the specific base sequence and a second primer having a sequence complementary to a part of the specific base sequence (herein, the first primer). Alternatively, either one of the second primers is added with an RNA polymerase promoter sequence at the 5 ′ end thereof, and the following steps (1) to (3) are performed.
(1) A first primer and a second primer in which a specific base sequence in Norovirus RNA is used as a template and a promoter sequence is added to either 5 ′ end, RNA-dependent DNA polymerase, ribonuclease H (RNase H), And a step of generating a double-stranded DNA containing a promoter sequence and the specific base sequence downstream of the promoter sequence by a DNA-dependent DNA polymerase,
(2) A step of generating an RNA transcript having the specific base sequence or its complementary sequence by RNA polymerase using the double-stranded DNA as a template,
(3) A step of amplifying the RNA transcript in a chain manner by using the RNA transcript as a template for the double-stranded DNA synthesis.

  The aforementioned amplification step has an enzyme having RNA-dependent DNA polymerase activity (reverse transcriptase) using single-stranded RNA as a template, an enzyme having RNase H activity, and a DNA-dependent DNA polymerase activity using single-stranded DNA as a template. Proceeds with enzymes and enzymes with RNA polymerase activity. As these enzymes, enzymes having several activities may be used, or a plurality of enzymes having respective activities may be used. For example, reverse transcriptase having three activities of RNA-dependent DNA polymerase activity, RNase H activity using single-stranded RNA as a template, and DNA-dependent DNA polymerase activity using single-stranded DNA as a template, and double-stranded DNA as a template It can be exemplified that it is used in combination with an RNA synthetase. However, an enzyme having RNase H activity may be further added to the reverse transcriptase and RNA synthase having these three activities as necessary. Examples of the above-mentioned reverse transcriptase having the three activities include AMV reverse transcriptase, MMLV reverse transcriptase, HIV reverse transcriptase and derivatives thereof, and among them, AMV reverse transcriptase and derivatives thereof are particularly preferable. Examples of the enzyme having RNA polymerase activity include T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase derived from bacteriophage widely used in molecular biology experiments and derivatives thereof.

  In order to proceed with the amplification step described above, in addition to the sample and each enzyme, a buffer, magnesium salt, potassium salt, nucleoside-triphosphate and ribonucleoside-triphosphate are added, and if necessary In order to adjust the reaction efficiency, dimethyl sulfoxide (DMSO), dithiothreitol (DTT), bovine serum albumin (BSA), sugar and the like are added, and the enzyme reaction is allowed to proceed under appropriate conditions. For example, when using AMV reverse transcriptase and T7 RNA polymerase, the reaction temperature may be set in the range of 35 to 65 ° C, preferably in the range of 40 ° C to 50 ° C.

  In the amplification step described above, when a promoter sequence is added to the first primer, the RNA transcript contains a sequence homologous to the template RNA, and when a promoter sequence is added to the second primer, RNA The transcript will contain the complementary sequence of the template RNA. The promoter sequence may be any sequence that can bind to RNA polymerase and initiate transcription, and known promoter sequences specific for various RNA polymerases can be used. For example, promoter sequences usually used in molecular biological experiments such as T7 promoter, SP6 promoter, T3 promoter and the like can be used without particular limitation. In addition to the promoter sequence, an additional sequence related to transcription efficiency such as an enhancer sequence may be further included.

When performing the amplification step described above, a promoter sequence may be added to either the first primer or the second primer, but when adding a promoter sequence to the first primer, the second primer And before or simultaneously with the step of synthesizing cDNA complementary to the specific nucleotide sequence using Norovirus RNA as a template by the enzyme having RNA-dependent DNA polymerase activity (step (1) described above) A cleaving oligonucleotide that overlaps with the 5 ′ end side of the homologous region with the first primer in the base sequence and that is complementary to the region adjacent to the 5 ′ end side from the overlapping site, and an enzyme having RNase H activity It is preferable to perform the step of cleaving the 5 ′ end side of the specific base sequence. By cleaving at the 5 ′ end of the specific nucleotide sequence in this way, after cDNA synthesis, the DNA strand complementary to the promoter sequence of the first primer hybridized to the cDNA is extended, and the 3 ′ end of the cDNA is extended. It is because it can synthesize | combine efficiently and can form a functional double stranded DNA promoter structure efficiently as a result. The cleavage method is not particularly limited as long as the site can be specifically cleaved, but it overlaps with the 5 ′ end site of the specific base sequence in Norovirus RNA (the partial sequence including the 5 ′ end site in the specific base sequence), In addition, an oligonucleotide DNA having a sequence complementary to a region adjacent to the 5 ′ direction (hereinafter referred to as “cleaving oligonucleotide DNA”) is added to form an RNA-DNA double strand, and the double strand A method of cleaving the RNA portion with an enzyme having ribonuclease H (RNase H) activity is preferred from the viewpoint of cleavage specificity and simplicity. In addition, the hydroxyl group at the 3 ′ end of the cleavage oligonucleotide DNA is preferably subjected to appropriate modification such as amination in order to prevent the elongation reaction.
As an oligonucleotide that can be detected rapidly and with high sensitivity to a wide range of genotypes of Norovirus GI RNA and Norovirus GII RNA, an oligonucleotide having a sequence of SEQ ID NOS: 12 and 13 as a first primer, Examples of the oligonucleotides whose primers are SEQ ID NOs: 14 to 16 and oligonucleotides for cleavage include oligonucleotides having the sequences described in SEQ ID NOs: 17 to 20.

The detection step comprises a step of measuring the amount of RNA transcript amplified in the amplification step using an oligonucleotide probe. Norovirus GI RNA present in the sample of the present invention and an oligonucleotide probe for identifying and detecting Norovirus GII RNA are 15 consecutive nucleotides in the sequence described in SEQ ID NO: 1 (Oligonucleotide probe 1). The oligonucleotide probe consisting of 15 or more consecutive bases in the sequence described in SEQ ID NO: 2 (Oligonucleotide probe 2) or its complementary sequence, or a complementary sequence thereof.
Moreover, as the oligonucleotide probe 1 described in SEQ ID NO: 1, any of the sequences described in SEQ ID NO: 3, 4, 5, 6 or their complementary sequences, and the oligonucleotide probe 2 described in SEQ ID NO: 2 As an example, an oligonucleotide probe comprising any one of the sequences shown in SEQ ID NOs: 7, 8, 9, 10, and 11 or a complementary sequence thereof can be exemplified. More preferably, an oligonucleotide probe comprising the sequence shown in SEQ ID NO: 5 or its complementary sequence, and the sequence shown in SEQ ID NO: 10 or its complementary sequence can be exemplified.
Also included in the probe of the present invention are oligonucleotide probes comprising sequences that can hybridize to the probe under stringent conditions. Stringent conditions can be selected from known conditions and are not particularly limited. For example, 50% (v / v) formamide at 42 ° C., 0.1% bovine serum albumin, 0.1% It means that it can hybridize under conditions where Ficoll, 0.1% polyvinyl pyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 150 mM sodium chloride, 75 mM sodium citrate and the like coexist.

  The detection using the oligonucleotide probe of the present invention can be performed by a method using electrophoresis or liquid chromatography, for example. In addition, for example, the probe may be bound to a detectable labeling substance and detected by a hybridization method. As the labeling substance, known substances such as enzymes, fluorescent dyes, radioisotopes and luminescent dyes can be used. In addition, molecular beacon (US Pat. No. 5,925,517, US Pat. No. 6,103,476), TaqMan probe (US Pat. No. 5210015, US Pat. No. 5,487,972), Q-Probe (US Pat. No. 3437816), cycling probe (US) Patents 5011769 and US Pat. No. 5,403,711) and intercalating fluorescent dye-labeled probes (see Patent Document 7 and Non-Patent Document 2) can be exemplified as preferred oligonucleotide probes of the present invention.

  Among them, when labeled with an intercalating fluorescent dye and forms a complementary double strand with a target nucleic acid, the fluorescence characteristics are changed by intercalating the intercalating fluorescent dye portion into the complementary double strand portion. Since the designed oligonucleotide probe can be detected in the course of the amplification process described above, the amplification process can be carried out by simply putting it in a container together with the reagents including the oligonucleotide DNA, primers, enzyme, enzyme substrate, etc. This is particularly preferable because the detection step can be performed. In the particularly preferred embodiment, the present invention can be carried out rapidly only by the operation of previously putting the above-described reagent or the like into a container and dispensing a predetermined amount of sample, and for example, a signal emitted from a fluorescent dye. If a part of the container is made of a transparent material so that it can be detected from the outside, the container can be sealed after dispensing the sample to prevent contamination between the samples.

The intercalating fluorescent dye is not particularly limited, and examples thereof include cyanine dyes such as oxazole yellow and thiazole orange, azo dyes such as hemicyanine dye, ethidium bromide, and methyl red, or derivatives thereof. For example, oxazole yellow is a dye that significantly increases fluorescence at 510 nm (excitation wavelength: 470 nm) when intercalated into double-stranded DNA. Such a dye may be bound to the oligonucleotide via an appropriate linker at the end of the oligonucleotide probe, the phosphodiester part or the base part. Further, in order to distinguish and detect two or more kinds of RNA amplification products, different dyes may be used for the respective oligonucleotide probes.
In addition, when detecting in the process of an amplification process, it is preferable that the oligonucleotide modifies the said hydroxyl group in order to prevent the extension | extension from the hydroxyl group of the 3 'terminal.

  According to the present invention, it is possible to identify and detect sequences specific to Norovirus GI RNA and Norovirus GII RNA, respectively. In addition, since this oligonucleotide probe set is difficult to form higher-order structures such as dimers and loops even at relatively low temperatures of 40 ° C. to 50 ° C., an amplification method that specifically amplifies RNA that does not require temperature change is provided. It is particularly effective when an amplification step is carried out and a specific base sequence is detected in the process.

  As an oligonucleotide probe used in the present invention, an amplification step is performed in the presence of an oligonucleotide labeled with an intercalating fluorescent dye, and if fluorescence intensity is detected over time in the process, any increase in fluorescence is recognized. The detection can be completed within this time, and norovirus RNA detection can be completed in about 20 minutes even if the time required for amplification is added.

  As described above, according to the present invention, it is possible to rapidly detect Norovirus GI RNA and Norovirus GII RNA in a sample in a rapid, sensitive and specific manner.

  Hereinafter, embodiments of the present invention will be described by way of examples. However, the present invention is not limited to these examples.

Example 1 Preparation of Oligonucleotide Labeled with Intercalating Fluorescent Dye Oligonucleotide Probe Labeled with Intercalating Fluorescent Dye (hereinafter referred to as “INAF Probe”) shown in (A) to (I) below Was prepared with reference to the method described in Non-Patent Document 3.
INAF probe (A): obtained by labeling thiazole orange with a linker on the phosphodiester moiety between the second thymine and the third adenine from the 5 ′ end in the sequence shown in SEQ ID NO: 3. An oligonucleotide probe for GI detection.
INAF probe (B): obtained by labeling thiazole orange with a linker on the phosphodiester moiety between the 15th thymine and the 16th adenine from the 5 ′ end in the sequence shown in SEQ ID NO: 4. An oligonucleotide probe for GI detection.
INAF probe (C): obtained by labeling thiazole orange with a linker on the phosphodiester moiety between the 14th thymine and the 15th adenine from the 5 ′ end of the sequence shown in SEQ ID NO: 5. An oligonucleotide probe for GI detection.
INAF probe (D): obtained by labeling thiazole orange with a linker on the phosphodiester moiety between the 15th thymine and the 16th adenine from the 5 ′ end in the sequence shown in SEQ ID NO: 6. An oligonucleotide probe for GI detection.
INAF probe (E): In the sequence shown in SEQ ID NO: 7, the phosphodiester moiety between the 12th cytosine and the 13th adenine from the 5 ′ end is labeled with the formula (1) via a linker. An oligonucleotide probe for GII detection obtained in the above.
INAF probe (F): In the sequence shown in SEQ ID NO: 8, the phosphodiester moiety between the 16th cytosine and the 17th adenine from the 5 ′ end is labeled with the formula (1) via a linker. An oligonucleotide probe for GII detection obtained in the above.
INAF probe (G): Formula (1) is labeled via a linker on the phosphodiester part between the second cytosine and the third guanine from the 5 ′ end in the sequence shown in SEQ ID NO: 9. An oligonucleotide probe for GII detection obtained in the above.
INAF probe (H): Formula (1) is labeled on the phosphodiester moiety between the 14th cytosine and the 15th adenine from the 5 ′ end of the sequence shown in SEQ ID NO: 10 via a linker. An oligonucleotide probe for GII detection obtained in the above.
INAF probe (I): Formula (1) is labeled on the phosphodiester moiety between the 14th cytosine and the 15th adenine from the 5 ′ end of the sequence shown in SEQ ID NO: 11 via a linker. An oligonucleotide probe for GII detection obtained in the above.

実施例２ 標準ＲＮＡの調製
後述の実施例で使用したノロウイルスＲＮＡ（以降、標準ＲＮＡと表記）は（１）から（２）に示す方法で調製した。
（１）ＧｅｎＢａｎｋに登録されているノロウイルスｃＤＮＡ配列のうち、表１に示す遺伝子型、および塩基配列領域の２本鎖ＤＮＡを調製した（なお、該ＤＮＡの５’末端側にはＳＰ６プロモーターを付加している）。
（２）（１）で調製したＤＮＡを鋳型として、ＳＰ６ ＲＮＡポリメラーゼを用いてインビトロ転写を実施し、引き続きＤＮａｓｅ Ｉ処理により前記２本鎖ＤＮＡを完全消化した後、ＲＮＡを精製して調製した。該ＲＮＡは２６０ｎｍにおける吸光度を測定して定量した。

Example 2 Preparation of Standard RNA Norovirus RNA (hereinafter referred to as standard RNA) used in Examples described later was prepared by the method shown in (1) to (2).
(1) Among the norovirus cDNA sequences registered in GenBank, double-stranded DNAs having the genotypes and nucleotide sequences shown in Table 1 were prepared (in addition, the SP6 promoter was added to the 5 ′ end of the DNA) doing).
(2) Using the DNA prepared in (1) as a template, in vitro transcription was performed using SP6 RNA polymerase. Subsequently, the double-stranded DNA was completely digested by DNase I treatment, and then RNA was purified and prepared. The RNA was quantified by measuring the absorbance at 260 nm.

実施例３ ノロウイルスＲＮＡ検出用オリゴヌクレオチドの評価
実施例１で作製したＩＮＡＦプローブを用いて、以下に示す方法で、検出性能および特異性について評価を行った。

Example 3 Evaluation of Norovirus RNA Detection Oligonucleotides Using the INAF probe prepared in Example 1, the detection performance and specificity were evaluated by the following method.

  (1) A reaction solution having the following composition was prepared.

Composition of the reaction solution (final concentration):
60 mM Tris-HCl buffer (pH 8.35)
300 mM trehalose 0.30 mM each dATP, dCTP, dGTP, dTTP
Each 2. 1 mM ATP, CTP, UTP
1.5 mM GTP
3.4 mM ITP
0.8 μM first primer 1 (T7 promoter added to the 5 ′ end side of the oligonucleotide consisting of the base sequence of SEQ ID NO: 12)
0.8 μM first primer 2 (T7 promoter added to the 5 ′ end side of the oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 13)
0.5 μM second primer 1 (SEQ ID NO: 14)
0.5 μM second primer 2 (SEQ ID NO: 15)
0.2 μM second primer 3 (SEQ ID NO: 16)
0.32 μM Cleavage oligonucleotide 1 (SEQ ID NO: 17)
0.32 μM Oligonucleotide 2 for cleavage (SEQ ID NO: 18)
0.16 μM Cleavage oligonucleotide 3 (SEQ ID NO: 19)
0.16 μM Cleavage oligonucleotide 4 (SEQ ID NO: 20)
60 nM INAF probe (prepared in Example 1)
0.025 mg / mL bovine serum albumin 142 U T7 RNA polymerase 6.4 U AMV reverse transcriptase.

(2) In order to evaluate the detection performance, the standard RNA prepared in Example 2 was added at 400 copies / test to the above reaction solution.
Moreover, in order to evaluate specificity, the standard RNA sample shown in Table 2 was added to the above reaction solution so as to be 4 × 10 ⁶ copies / test. Thereafter, the mixture was kept at 46 ° C. for 5 minutes, and then an initiator solution having the following composition was added and stirred.

Starting solution composition (final concentration):
9.5% dimethyl sulfoxide 19 mM magnesium chloride 95 mM potassium chloride.

  (3) Subsequently, the tube to which each reagent is added is applied to a fluorescence spectrophotometer with a temperature control function capable of directly detecting the reaction, and the reaction solution is reacted at 46 ° C. , Or 545 nm) was measured over time for 20 minutes.

  When the start solution is added and stirring is completed, the reaction time is 0 minute, and the reaction solution fluorescence intensity ratio (the value obtained by dividing the fluorescence intensity value for a given time by the background fluorescence intensity ratio) exceeds 2.0. And the time at that time was defined as the detection time. The results are shown in Tables 2 and 3. “ND” in Table 3 means that the fluorescence intensity ratio 20 minutes after the start of the reaction was 2.0 or less (negative judgment).

  As for the detection performance of Norovirus RNA (Table 2), the oligonucleotide probes for GI detection and GII detection examined in this Example both had 400 copies / test of Norovirus GI RNA or Norovirus GII RNA within 20 minutes. Detected. In particular, the oligonucleotide probes of SEQ ID NOs: 3 to 10 detect 400 copies / test of norovirus RNA within 10 minutes, and can be said to be oligonucleotide probes capable of rapidly detecting norovirus RNA.

特異性（表３）については、本実施例で検討したＧＩ検出用オリゴヌクレオチドプローブいずれもが４×１０^６コピー／ｔｅｓｔのＧＩＩ標準ＲＮＡを検出せず、ノロウイルス ＧＩ ＲＮＡに対して高い特異性を有しているといえる。同様に、本実施例で検討したＧＩＩ検出用オリゴヌクレオチドプローブいずれもが４×１０^６コピー／ｔｅｓｔのＧＩ標準ＲＮＡを検出せず、ノロウイルス ＧＩＩ ＲＮＡに対して高い特異性を有しているといえる。

Regarding the specificity (Table 3), none of the GI detection oligonucleotide probes examined in this example detected 4 × 10 ⁶ copies / test GII standard RNA, and high specificity for Norovirus GI RNA. It can be said that it has. Similarly, none of the GII detection oligonucleotide probes examined in this example detected GI standard RNA of 4 × 10 ⁶ copies / test and can be said to have high specificity for Norovirus GII RNA. .

実施例４ ノロウイルス ＧＩ ＲＮＡ、および、ノロウイルス ＧＩＩ ＲＮＡの同時検出
実施例１で作製したＩＮＡＦプローブを用いて、ノロウイルスＲＮＡのＧＩ、および、ＧＩＩを同時に識別して検出することが可能か以下に示す方法で、評価を行った。

Example 4 Simultaneous detection of Norovirus GI RNA and Norovirus GII RNA Whether the GI and GII of Norovirus RNA can be simultaneously identified and detected using the INAF probe prepared in Example 1 is the following method: And evaluated.

  (1) A reaction solution having the following composition was prepared.

Composition of the reaction solution (final concentration):
60 mM Tris-HCl buffer (pH 8.35)
300 mM trehalose 0.30 mM each dATP, dCTP, dGTP, dTTP
Each 2. 1 mM ATP, CTP, UTP
1.5 mM GTP
3.4 mM ITP
0.8 μM first primer 1 (T7 promoter added to the 5 ′ end side of the oligonucleotide consisting of the base sequence of SEQ ID NO: 12)
0.8 μM First primer 2 (T7 promoter (sequence number added) to the 5 ′ end of the oligonucleotide consisting of the base sequence of SEQ ID NO: 13)
0.5 μM second primer 1 (SEQ ID NO: 14)
0.5 μM second primer 2 (SEQ ID NO: 15)
0.2 μM second primer 3 (SEQ ID NO: 16)
0.32 μM Cleavage oligonucleotide 1 (SEQ ID NO: 17)
0.32 μM Oligonucleotide 2 for cleavage (SEQ ID NO: 18)
0.16 μM Cleavage oligonucleotide 3 (SEQ ID NO: 19)
0.16 μM Cleavage oligonucleotide 4 (SEQ ID NO: 20)
60 nM GI detection INAF probe (SEQ ID NO: 5)
60 nM GII detection INAF probe (SEQ ID NO: 10)
0.025 mg / mL bovine serum albumin 142 U T7 RNA polymerase 6.4 U AMV reverse transcriptase.

  (2) The standard RNA sample shown in Table 4 was added to the above reaction reagent so as to be 400 copies / test. However, for “GI.6 + GII.4”, GI. 6 and GII. 4 and 400 copies / test were added. Then, it kept at 46 degreeC for 5 minute (s), Then, the starting liquid which consists of the following compositions was added and stirred.

Starting solution composition (final concentration):
9.5% dimethyl sulfoxide 19 mM magnesium chloride 95 mM potassium chloride.

  (3) Subsequently, the tube to which each reagent is added is applied to a fluorescence spectrophotometer with a temperature control function capable of directly detecting the reaction, and the reaction solution is reacted at 46 ° C. , Or 545 nm) was measured over time for 20 minutes.

  When the start solution is added and stirring is completed, the reaction time is 0 minute, and the reaction solution fluorescence intensity ratio (the value obtained by dividing the fluorescence intensity value for a given time by the background fluorescence intensity ratio) exceeds 2.0. And the time at that time was defined as the detection time. The results are shown in Table 4. “ND” in Table 4 means that the fluorescence intensity ratio 20 minutes after the start of the reaction was 2.0 or less (negative judgment).

  For simultaneous detection of Norovirus GI RNA and Norovirus GII RNA (Table 4), it was confirmed that Norovirus GI RNA and Norovirus GII RNA can be detected separately in a single sealed container and at a constant temperature.

Claims (3)

A method for distinguishing and detecting genogroup I and genogroup II of norovirus RNA present in a sample, comprising:
(1) a step of amplifying a specific base sequence capable of distinguishing between genogroup I and genogroup II in norovirus RNA, and (2) a step of detecting the amplified sequence,
Have
The method according to claim 1, wherein the amplifying step and the detecting step are performed in a single sealed container and at a constant temperature. The method of claim 1, wherein the detecting step is performed by real-time monitoring. (1) A first primer and a second primer having a specific base sequence in Norovirus RNA as a template and a promoter sequence added to either 5 ′ end, RNA-dependent DNA polymerase, ribonuclease H (RNase H), and A step of generating a double-stranded DNA comprising a promoter sequence and the specific base sequence downstream of the promoter sequence by a DNA-dependent DNA polymerase;
(2) A step of generating an RNA transcript having the specific base sequence or its complementary sequence by RNA polymerase using the double-stranded DNA as a template,
(3) a step of amplifying the RNA transcript in a chain manner by using the RNA transcript as a template for the subsequent double-stranded DNA synthesis;
(4) measuring the amount of the RNA transcript,
And the step (4) of measuring the amount of RNA transcript in the presence of a nucleic acid probe designed to change the signal property when a complementary double strand is formed with the target RNA. The nucleic acid probe is made by measuring a change, and the nucleic acid probe is an oligonucleotide probe consisting of 15 or more consecutive bases in the sequence shown in SEQ ID NO: 1 or its complementary sequence, and a continuous in the sequence shown in SEQ ID NO: 2. The method according to claim 1 or 2, which is an oligonucleotide probe comprising 15 or more bases or a complementary sequence thereof.