JP2009017824A - Improved method for detecting norovirus rna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To detect a wide range of gene types of Norovirus RNA with high sensitivity. <P>SOLUTION: The amount of an RNA product amplified in the RNA amplification process is measured by a nucleic acid probe labeled with an intercalator fluorescent dye for a wide range of gene types of Norovirus RNA, wherein the RNA amplification process comprises steps for generating double-stranded DNA containing a promotor sequence with ≥2 kinds of cutting oligonucleotides having high hybridization efficiency, a first primer having a promotor sequence at a 5'-terminal, a second primer and a reverse transcription enzyme, subsequently generating RNA transcription product with RNA polymerase with the double-stranded DNA as a template and generating the double-stranded DNA with the RNA transcription product succeedingly as a template of the DNA synthesis with the reverse transcription enzyme. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for easily and rapidly measuring Norovirus. The present invention is useful in the fields of clinical testing, public health, food testing, and food poisoning testing.

  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).

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 has been detected in infants (sporadic) acute gastroenteritis, and cases of human-to-human infection have been reported. From the above, norovirus testing has become a major issue in public health and food quality control, and it is a highly sensitive, rapid, and capable of detecting all genotypes using a gene amplification method, or a high detection rate test. The development of a law is desired. Conventionally, norovirus detection has been based on observation with an electron microscope. Although all genotypes can be detected by this method, the amount of virus of 10 ⁶ cells / mL or more is required for detection and the sensitivity is low, so the specimen is limited to patient stool. Moreover, even if the virus could be observed, it could not be identified.

  A reagent for specific antibody detection ELISA using virus-like hollow particles of human calicivirus has also been developed (Patent Document 1). However, the detection sensitivity is similar to that of an electron microscope, and it cannot be said that the sensitivity is high.

  As a means for measuring norovirus with high sensitivity, there is a method of amplifying norovirus RNA by RT-PCR (Patent Document 2). In this method, generally, there are two steps: a reverse transcription (RT) step and a PCR step. This process is necessary. This not only becomes a factor of complicating operations and deteriorating reproducibility, but also increases the risk of secondary contamination. In addition, when the RT process and the PCR process are combined, it usually takes 2 hours or more, which is not suitable 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. 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, NASBA methods (Patent Documents 3 and 4) and TMA method (Patent Document 5) have been reported as methods for amplifying only RNA at a constant temperature. 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.

  Examples of a method for easily amplifying and measuring RNA include the method of Ishiguro et al. (Patent Document 6 and Non-Patent Document 2). 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 fluorescence characteristics is measured. RNA amplification and measurement is performed in a simple, constant temperature, one-step, sealed container. Can be carried out simultaneously.

  Each of the above nucleic acid amplification methods is a method of amplifying a target RNA using a primer set consisting of a sense primer (first primer) and an antisense primer (second primer), and a primer sequence constituting the primer set It is well known that these combinations have important implications for the efficiency and specificity of nucleic acid amplification. However, since norovirus has extremely diverse genotypes, it has been extremely difficult to construct a primer set that uniformly and efficiently amplifies all genotypes.

WO2000 / 079280 Japanese Patent No. 3752102 Japanese Patent No. 2650159 Japanese Patent No. 3152927 Japanese Patent No. 3241717 JP 2000-14400 A JP 2005-245434 A Japanese Patent Laid-Open No. 8-211050 JP 2001-13147 A Kageyama T. et al. , Journal of Clinical Microbiology, 41, 1548-1557 (2003). Ishiguro T. et al. , Analytical Biochemistry, 314, 77-86 (2003). Infectious Disease Development Trend Survey Weekly Report, 6 (11), 14-19 (2004) Ishiguro T. et al. , Nucleic Acids Research, 24, 4992-4997 (1996).

  Norovirus is roughly divided into two gene groups, Genogroup I (GI) and Genogroup II (GII), depending on the difference in gene sequence. Further, for each gene group, GI is currently classified into 14 genotypes and GII is differentiated into 17 genotypes (see Non-Patent Document 3). The base sequence homology between genotypes belonging to GI and between genotypes belonging to GII is about 70%. The homology between the base sequences of GI and GII is 40 to 50%.

  In order to increase the detection rate with the norovirus detection reagent using the gene amplification method, at least two regions of 20 bases or more in which the base sequence is highly conserved among various genotypes are required as primer binding regions. It is. However, the following 6 sequences (Chiba (No. AB042808), Norwalk (No. M87661), Southampton (No. L07418), Chamberwell (No. AF145896), Hawaii (No. U07611), as the sequence of Norovirus on GenBank. Even by examining the homology of HuCV (No. AY032605)), there is no region of 20 bases or more having the same base sequence between genotypes.

  Under such a background, the present inventors have already provided a detection method having a high detection rate with a wide genotype of GI or GII (Japanese Patent Laid-Open No. 2005-245434, Patent Document 7). . However, even with this method, it cannot be said that highly sensitive and uniform detection for all genotypes of Norovirus has been achieved, and a method for detecting Norovirus RNA that detects a wider range of genotypes with higher sensitivity is desired. ing.

  As a result of intensive studies to solve the above problems, the present inventor has made it possible to perform simple and rapid measurement with high sensitivity to a wide range of genotypes of Norovirus RNA.

The invention according to claim 1 is a method for detecting Norovirus RNA in a sample, comprising:
(1) Using a specific nucleic acid sequence in Norovirus RNA as a template, overlapping with the 5 ′ terminal region of the homologous region with the first primer in the specific nucleotide sequence, and complementary to a region adjacent to the 5 ′ direction from the site Cleaving the RNA at the 5 ′ end site of the specific base sequence with a cleavable oligonucleotide and ribonuclease H (RNase H),
(2) The first base, the second primer, and the RNA-dependent DNA polymerase, RNase H, and DNA-dependent DNA polymerase with a promoter sequence added to the 5 ′ end, and the specific base downstream of the promoter sequence Generating a double-stranded DNA comprising the sequence;
(3) generating an RNA transcript comprising the specific base sequence by RNA polymerase using the double-stranded DNA as a template;
(4) 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;
(5) measuring the amount of the RNA transcript,
And the cleavage oligonucleotide comprises at least two kinds of oligonucleotides selected from oligonucleotides each having a sufficiently complementary sequence to SEQ ID NOs: 1 to 3, and the first primer is SEQ ID NO: 4 Wherein the second primer comprises a sequence sufficiently complementary to SEQ ID NO: 5.

  The invention according to claim 2 relates to claim 1, wherein in the method of detecting norovirus RNA, the step (5) of measuring the amount of RNA transcript produces signal characteristics when a complementary double strand is formed with the target RNA. It is made by measuring the signal change in the presence of a nucleic acid probe designed to change, and the nucleic acid probe consists of a sequence sufficiently complementary to SEQ ID NO: 6.

  The present invention is described in detail below.

  The specific base sequence in the present invention is an RNA or DNA base homologous to the base sequence from the 5 ′ end of the homologous region of the first primer to the 3 ′ end of the complementary region of the second primer on the norovirus RNA. Indicates the sequence. That is, in the present invention, an RNA transcript derived from the specific base sequence is amplified. The 5 ′ end site of the homologous region with the first primer in the present invention comprises a partial sequence containing the 5 ′ end of the homologous region within the specific base sequence, and this site is a complementary region with the cleavage oligonucleotide. And a region where homologous regions of the first primer overlap. The promoter in the present invention is a promoter sequence specific to various RNA polymerases at the site where RNA polymerase binds and initiates transcription, and is not particularly limited in the use of the present invention. The T7 promoter, SP6 promoter, T3 promoter and the like that are widely used in scientific experiments and the like are suitable.

  A sufficiently complementary sequence in the present invention can be specifically and efficiently hybridized to a specific base sequence under optimized nucleic acid amplification reaction conditions (salt concentration, oligonucleotide concentration, reaction temperature, etc.). An array. In addition, a sufficiently homologous sequence in the present invention means a specific and highly specific sequence for a completely complementary sequence of a specific base sequence under optimized nucleic acid amplification reaction conditions (salt concentration, oligonucleotide concentration, reaction temperature, etc.). A sequence that can be efficiently hybridized. Therefore, the length and the like of the sufficiently complementary or sufficiently homologous sequences referred to in the present invention can be arbitrarily set as long as they do not affect the specificity and efficiency of hybridization.

  In the present invention, Norovirus RNA is cleaved at the 5 'end site of a specific nucleic acid sequence in the RNA before becoming a template for cDNA synthesis. By cleaving at the 5 ′ end site of the specific nucleic acid sequence, after cDNA synthesis, a DNA strand complementary to the promoter sequence of the first primer hybridized to the cDNA is extended by extending the 3 ′ end of the cDNA. It can be synthesized efficiently and results in the formation of a functional double stranded DNA promoter structure. As such a cleavage method, it is complementary to a region adjacent to the 5 ′ direction overlapping with the 5 ′ end site of the specific base sequence in Norovirus RNA (partial sequence including the 5 ′ end in the specific base sequence). And a method of cleaving the RNA portion of the RNA-DNA hybrid formed by adding an oligonucleotide having a proper sequence (hereinafter referred to as a cleaving oligonucleotide) with an enzyme having ribonuclease H (RNase H) activity. It is preferable to use a hydroxyl group at the 3 'end of the cleaving oligonucleotide that has been appropriately modified to prevent an extension reaction, such as an amino group. In order to cleave the 5 'end site with high efficiency, it is necessary to increase the hybridization efficiency of the cleaving oligonucleotide. However, since norovirus RNA has various genotypes, it is difficult to efficiently hybridize to various genotypes with one type of cleavage oligonucleotide. Therefore, in the present invention, at least two kinds of oligonucleotides selected from cleavage oligonucleotides each consisting of a sequence sufficiently complementary to the sequences described in SEQ ID NOs: 1 to 3 are used as the cleavage oligonucleotides. Improves the hybridization efficiency of the cleaving oligonucleotide for a wider range of genotypes of Norovirus RNA, resulting in a wider range of the first and second primers used Sensitivity for various genotypes has become possible.

  The target RNA in the present invention refers to a sequence other than the homologous or complementary region of the primer among the specific base sequence on the RNA transcript, and complementary binding to a nucleic acid probe labeled with an intercalating fluorescent dye. Has a sequence that is possible. Therefore, the nucleic acid probe labeled with the intercalating fluorescent dye has a sequence complementary to a part of the specific base sequence in the present invention. Therefore, for example, as an embodiment of the present invention, a nucleic acid probe labeled with an intercalating fluorescent dye can be a nucleic acid probe having a sufficiently complementary sequence to SEQ ID NO: 6.

  As one aspect of the present invention, the cleaving oligonucleotide has a sequence that is sufficiently complementary to SEQ ID NOs: 1 to 3, respectively, and is sufficiently complementary to Norovirus RNA. The first primer has a promoter sequence at its 5 ′ end, has a sequence sufficiently homologous to SEQ ID NO: 4, and is sufficiently complementary to the fully complementary sequence of Norovirus RNA. Oligonucleotide. The second primer has a sequence sufficiently complementary to SEQ ID NO: 5 and is sufficiently complementary to Norovirus RNA. Furthermore, the nucleic acid probe labeled with the intercalating fluorescent dye preferably has a sufficiently complementary sequence to SEQ ID NO: 6 and is sufficiently complementary to the target nucleic acid.

  More preferably, as the cleavage oligonucleotide of the present invention, the oligonucleotide sequence shown in SEQ ID NOs: 7 to 9, the first primer is the oligonucleotide sequence shown in SEQ ID NO: 4, and the second primer is SEQ ID NO: The oligonucleotide sequence described in 10 and the nucleic acid probe labeled with the intercalating fluorescent dye preferably have the oligonucleotide sequence described in SEQ ID NO: 11.

  In the method for measuring Norovirus RNA in the present invention, each enzyme (an enzyme having RNA-dependent DNA polymerase activity (reverse transcriptase) using single-stranded RNA as a template (reverse transcriptase), an enzyme having RNase H activity, and single-stranded DNA as a template) An enzyme having a DNA-dependent DNA polymerase activity and an enzyme having an RNA polymerase activity). As each enzyme, an enzyme having several activities may be used, or a plurality of enzymes having respective activities may be used. In addition, for example, a reverse transcriptase having both 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, has RNA polymerase activity. It is possible not only to add the enzyme having, but also to supplement with additional enzyme having RNase H activity if necessary. The reverse transcriptase is preferably AMV reverse transcriptase, MMLV reverse transcriptase, HIV reverse transcriptase, or a derivative thereof widely used in molecular biological experiments. Most preferred. In addition, as the enzyme having RNA polymerase activity, bacteriophage-derived T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and derivatives thereof, which are widely used in molecular biological experiments and the like, can be used.

  The cleaving oligonucleotide is added to the Norovirus RNA in the sample, and the RNA is cleaved at the 5 'end site of the specific base sequence by the RNase H activity of the reverse transcriptase. When a reverse transcription reaction with the reverse transcriptase is performed in the presence of the first primer and the second primer using the cleaved RNA as a template, the second primer binds to a specific base sequence in Norovirus RNA, CDNA synthesis is performed by the RNA-dependent DNA polymerase activity of the reverse transcriptase. In the obtained RNA-DNA hybrid, the RNA portion is decomposed by the RNase H activity of the reverse transcriptase and dissociated, whereby the first primer binds to the cDNA. Subsequently, double-stranded DNA derived from a specific base sequence and having a promoter sequence at the 5 'end is generated by the DNA-dependent DNA polymerase activity of the reverse transcriptase. The double-stranded DNA contains a specific base sequence downstream of the promoter sequence, and an RNA transcript derived from the specific base sequence is produced by the RNA polymerase. The RNA transcript becomes a template for the double-stranded DNA synthesis by the first and second primers, and a series of reactions proceed in a chain, and the RNA transcript is amplified.

  In order to proceed such a chain reaction, it is said that at least a buffer, a magnesium salt, a potassium salt, a nucleoside-triphosphate, and a ribonucleoside-triphosphate are included as known elements essential to each enzyme. Not too long. In addition, dimethyl sulfoxide (DMSO), dithiothreitol (DTT), bovine serum albumin (BSA), sugar, and the like can be added as additives for adjusting reaction efficiency.

  For example, when AMV reverse transcriptase and T7 RNA polymerase are used, the reaction temperature is preferably set in the range of 35 to 65 ° C, particularly preferably in the range of 40 to 44 ° C. The RNA amplification process proceeds at a constant temperature, and the reaction temperature can be set to any temperature at which reverse transcriptase and RNA polymerase exhibit activity.

  The amount of the amplified RNA transcript can be measured by a known nucleic acid measurement method. As such a measuring method, a method using electrophoresis or liquid chromatography, a hybridization method using a nucleic acid probe labeled with a detectable label, and the like can be used. However, these are multi-step operations, and the amplification products are taken out of the system for analysis, and therefore there is a great risk of scattering of amplification products to the environment causing secondary contamination. In order to overcome these problems, it is preferable to use a nucleic acid probe designed so that the fluorescence property is changed by complementary binding to the target nucleic acid. As a more preferred method, when the nucleic acid probe labeled with an intercalating fluorescent dye forms a complementary double strand with the target nucleic acid, the intercalating fluorescent dye portion intercalates with the complementary double strand portion. Thus, there is a method in which the nucleic acid amplification step is performed in the presence of a nucleic acid probe designed to change the fluorescence characteristics, and the change in the fluorescence characteristics is measured (see Patent Document 6 and Non-Patent Document 2).

  Although it does not specifically limit as said intercalator fluorescent dye, Oxazole yellow, thiazole orange, ethidium bromide, these derivatives, etc. which are used widely can be utilized. The change in the fluorescence characteristics includes a change in fluorescence intensity. For example, in the case of oxazole yellow, it is known that fluorescence at 510 nm (excitation wavelength: 490 nm) is significantly increased by intercalating with double-stranded DNA. The nucleic acid probe labeled with the intercalating fluorescent dye is an oligonucleotide that is sufficiently complementary to the RNA transcript, and is intercalating fluorescent via an appropriate linker at the end, phosphodiester part or base part. It has a structure in which a dye is bound and the hydroxyl group at the 3 ′ end is appropriately modified for the purpose of preventing extension from the hydroxyl group at the 3 ′ end (see Patent Document 8 and Non-Patent Document 4).

  As for the labeling of the intercalator fluorescent dye on the oligonucleotide, it is possible to introduce a functional group into the oligonucleotide and bind the intercalator fluorescent dye by a known method (see Patent Document 9 and Non-Patent Document 4). . In addition, as a method for introducing the functional group, it is also possible to use widely used Label-ON Reagents (manufactured by Clontech).

  As one embodiment of the present invention, the sample has at least a first primer having a T7 promoter sequence at the 5 ′ end (a sequence obtained by adding a T7 promoter sequence (SEQ ID NO: 13) to the 5 ′ end of the sequence described in SEQ ID NO: 4). , A second primer (sequence shown in SEQ ID NO: 10), a nucleic acid probe labeled with an intercalating fluorescent dye (sequence shown in SEQ ID NO: 11), a cleavage oligonucleotide (sequences shown in SEQ ID NO: 7 to 9 respectively), Amplification reagents including AMV reverse transcriptase, T7 RNA polymerase, buffer, magnesium salt, potassium salt, nucleoside-triphosphate, ribonucleoside-triphosphate, dimethyl sulfoxide (DMSO) were added, and the reaction temperature was 35 to 65 ° C. The reaction is carried out at a constant temperature (preferably 40 to 44 ° C.) and at the same time the fluorescence intensity of the reaction solution is changed over time. To provide a method for constant. In the above embodiment, since the fluorescence intensity is measured over time, the measurement can be completed at any time when a significant increase in fluorescence is observed, and the nucleic acid amplification and measurement are usually completed within one hour. Is possible.

  It should be noted that all the samples contained in the measurement reagent can be sealed in a single container. That is, as long as a certain amount of sample is dispensed into such a single container, norovirus RNA can be automatically amplified and detected thereafter. This container only needs to be made of a transparent material at least partially so that the signal emitted from the fluorescent dye can be measured from the outside, and any container that can be sealed after dispensing a sample is contaminated. It is particularly preferable in terms of preventing nations.

  Since the RNA amplification / measurement method of the above embodiment can be carried out at a constant temperature in one step, it can be said that it is a simpler method suitable for automation than RT-PCR. According to the present invention, high specificity, high sensitivity, rapidity, convenience, constant temperature and one-step measurement can be performed for a wide range of genotypes of norovirus RNA.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by these Examples.
Example 1
Norovirus RNA (hereinafter referred to as standard RNA) used in Examples of the present application was prepared by the method shown in (1) to (2).
(1) Among the norovirus cDNA base sequences registered in GenBank, double-stranded DNAs having the genotypes and base sequence regions shown in Table 1 were prepared (in addition, the SP6 promoter was placed on the 5 ′ end side of the DNA) Added).
(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.

なお、標準ＲＮＡの全長は５３３塩基（該ＲＮＡの５’末端にはＳＰ６プロモーターに由来する８塩基が付加されている）と、ノロウイルスＲＮＡの全長（約７０００塩基）の一部ではあるが、本発明の測定対象であるノロウイルスＲＮＡの測定には十分適用可能である。また、今回標準ＲＮＡを調製した遺伝子型はノロウイルス ＧＩＩ ＲＮＡの遺伝子型全１７種のうちの７種と一部ではあるが、
（Ａ）ＧＩＩ／７、ＧＩＩ／８、ＧＩＩ／９、およびＧＩＩ／１３との間
（Ｂ）ＧＩＩ／１１、ＧＩＩ／１４、およびＧＩＩ／１６との間
（Ｃ）ＧＩＩ／１、ＧＩＩ／１２、およびＧＩＩ／１５との間
（Ｄ）ＧＩＩ／２、ＧＩＩ／５、およびＧＩＩ／１０との間
はそれぞれ相同性が高い（非特許文献３）ことから、今回調製した遺伝子型の標準ＲＮＡを全て検出できれば、特殊な遺伝子型であるＧＩＩ／１７以外の全ての遺伝子型に属するノロウイルス ＧＩＩ ＲＮＡを検出可能であることが予想される。
実施例２
インターカレーター性蛍光色素で標識された核酸プローブを調製した。非特許文献４に記載の方法で、配列番号１１に記載の配列の５’末端から１２番目のＣと１３番目のＡの間のリン酸ジエステル部分にリンカーを介してオキサゾールイエローを結合させたオキサゾールイエロー標識核酸プローブを調製した（図１）。
実施例３
本願発明の方法において、表２に示す組み合わせの第一のプライマー、第二のプライマー、インターカレーター性蛍光色素で標識された核酸プローブ（以降、ＩＮＡＦプローブと表記）、切断用オリゴヌクレオチドを用いて、（１）から（４）に示す方法で、標準ＲＮＡの測定を行なった。なお、表２の組み合わせのうち、組み合わせＡに記載の第一のプライマー、第二のプライマー、切断用オリゴヌクレオチドは特開２００５−２４５４３４（特許文献７）の実施例１に開示した配列と同一である。

The total length of the standard RNA is 533 bases (8 bases derived from the SP6 promoter are added to the 5 ′ end of the RNA) and part of the total length of the norovirus RNA (about 7000 bases). The present invention is sufficiently applicable to the measurement of Norovirus RNA that is the measurement target of the invention. In addition, the genotype for which the standard RNA was prepared this time was a part of seven of the 17 genotypes of norovirus GII RNA.
(A) Between GII / 7, GII / 8, GII / 9, and GII / 13 (B) Between GII / 11, GII / 14, and GII / 16 (C) GII / 1, GII / 12 And GII / 15 (D) GII / 2, GII / 5, and GII / 10 are highly homologous to each other (Non-patent Document 3). If all can be detected, it is expected that Norovirus GII RNA belonging to all genotypes other than the special genotype GII / 17 can be detected.
Example 2
A nucleic acid probe labeled with an intercalating fluorescent dye was prepared. Oxazole obtained by binding oxazole yellow to a phosphodiester moiety between 12th C and 13th A from the 5 ′ end of the sequence shown in SEQ ID NO: 11 via a linker by the method described in Non-Patent Document 4 A yellow labeled nucleic acid probe was prepared (FIG. 1).
Example 3
In the method of the present invention, a first primer, a second primer, a nucleic acid probe labeled with an intercalating fluorescent dye (hereinafter referred to as an INAF probe), and a cleavage oligonucleotide, as shown in Table 2, Standard RNA was measured by the methods shown in (1) to (4). Of the combinations shown in Table 2, the first primer, the second primer, and the cleavage oligonucleotide described in combination A are the same as the sequences disclosed in Example 1 of JP-A-2005-245434 (Patent Document 7). is there.

（１）前記各標準ＲＮＡをＲＮＡ希釈液（１０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ８．０）、１．０ｍＭ ＥＤＴＡ、０．２５Ｕ／μＬ リボヌクレアーゼインヒビター、５．０ｍＭ ＤＴＴ）を用い１０^２または１０^３コピー／５μＬとなるように希釈した。
（２）以下の組成の反応液２０μＬを０．５ｍＬ容ＰＣＲチューブ（Ｉｎｄｉｖｉｄｕａｌ Ｄｏｍｅ Ｃａｐ ＰＣＲ Ｔｕｂｅ、ＳＳＩ製）に分注し、これに前記ＲＮＡ試料５μＬを添加した。

(1) 10 ² or 10 ³ copies of each standard RNA using an RNA diluent (10 mM Tris-HCl buffer (pH 8.0), 1.0 mM EDTA, 0.25 U / μL ribonuclease inhibitor, 5.0 mM DTT) Dilute to / 5 μL.
(2) 20 μL of the reaction solution having the following composition was dispensed into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and 5 μL of the RNA sample was added thereto.

Composition of reaction solution: final concentration or amount after addition of enzyme solution (in 30 μL) 60 mM Tris-HCl buffer (pH 8.6)
17.2 mM magnesium chloride 120 mM potassium chloride 1.0 mM DTT
0.25 mM dATP, dCTP, dGTP, dTTP each
Each 3.0 mM ATP, CTP, GTP, UTP
3.6 mM ITP
0.75 μM First primer: T7 promoter sequence (SEQ ID NO: 13) is added to the 5 ′ end of the base sequence described in each SEQ ID NO. 1.0 μM second primer 25 nM INAF probe: the probe Are prepared in Example 2. Each 0.16 μM cleavage oligonucleotide: hydroxyl group at the 3 ′ end of the oligonucleotide is modified with an amino group 6U ribonuclease inhibitor (manufactured by Takara Bio Inc.)
13% DMSO
(3) After the above reaction solution was kept at 43 ° C. for 5 minutes, 5 μL of enzyme solution having the following composition and kept at 43 ° C. for 2 minutes in advance was added.

Composition of enzyme solution: final concentration or amount at the time of reaction (in 30 μL) 2.0% sorbitol 6.4U AMV reverse transcriptase (Life Science)
142U T7 RNA polymerase (Invitrogen)
3.6 μg Bovine Serum Albumin (4) Subsequently, using a fluorescence spectrophotometer with a temperature control function capable of directly measuring a PCR tube, the reaction solution was reacted at 43 ° C. and simultaneously the fluorescence intensity of the reaction solution (excitation wavelength: 470 nm, fluorescence wavelength: 520 nm). Measurement was performed over time for 60 minutes.

The time when the enzyme is added is defined as positive when the fluorescence intensity ratio of the reaction liquid (the value obtained by dividing the fluorescence intensity value of the predetermined time by the fluorescence intensity value of the background) exceeds 1.2. the result of the detection time are shown in Table 3 (the initial RNA amount 10 ³ results for copying) and Table 4 (results of the initial RNA amount 10 ² copies). In Tables 3 and 4, N. D. Means a sample having a fluorescence intensity ratio of less than 1.2 (negative determination) 60 minutes after the enzyme was added.

以前、本発明者らが提供したノロウイルスの検出方法（特開２００５−２４５４３４号公報、特許文献７）では、実施例１において、本願表２の組み合わせＡに示す第一のプライマー、第二のプライマー、および切断用オリゴヌクレオチドを用いることでＧＩＩ／１（Ｈａｗａｉｉ）、ＧＩＩ／２（Ｓｏｕｔｈａｍｐｔｏｎ）、ＧＩＩ／３（Ｍｅｘｉｃｏ）、ＧＩＩ／４（Ｃａｍｂｅｒｗｅｌｌ）に属するノロウイルス ＧＩＩ 人工ＲＮＡ（本願実施例１で調製したものとは塩基長が異なる）１０^６コピーを検出したことを開示しているが、今回、初期ＲＮＡ量が低い条件で増幅・検出反応を行なったところ、遺伝子型に対する検出率は初期ＲＮＡ量１０^３コピーでは７種中２種、１０^２コピーでは７種全て未検出と、低濃度のノロウイルスＲＮＡに対する検出率が低いことが判明した。そこで、切断用オリゴヌクレオチドを配列番号７に変更（組み合わせＢ）したところ、遺伝子型に対する検出率は初期ＲＮＡ量１０^３コピーでは７種全て、１０^２コピーでは７種中１種検出と、低濃度のノロウイルスＲＮＡに対する検出率が向上した。さらに切断用オリゴヌクレオチドを二種以上使用すること（組み合わせＣからＥ）で初期ＲＮＡ量１０^２コピーでの遺伝子型に対する検出率はより向上し、最も好ましい組み合わせである組み合わせＥでの初期ＲＮＡ量１０^２コピーにおける遺伝子型に対する検出率は７種中５種まで向上した。

Previously, in the method for detecting norovirus provided by the present inventors (Japanese Patent Laid-Open No. 2005-245434, Patent Document 7), in Example 1, the first primer and the second primer shown in the combination A in Table 2 of this application And Norovirus GII artificial RNA belonging to GII / 1 (Hawaii), GII / 2 (Southampton), GII / 3 (Mexico), GII / 4 (Camberwell) by using a cleavage oligonucleotide (prepared in Example 1 of the present application) Although the base length as that discloses the detection of the different) 10 ⁶ copies, this, when the initial RNA amount was performed amplification-detection reaction at a low condition, the detection rate for genotype initial RNA amount 10 ³ 2 kinds of seven a copy, and all seven undetected at 10 ² copies, low concentrations of Norouiru The low detection rate for the RNA has been found. Therefore, when the oligonucleotide for cleaving was changed to SEQ ID NO: 7 (combination B), the detection rate for the genotype was as low as 10% for the initial RNA amount, all 7 types for 10 ³ copies, and 1 for 7 types for 10 ² copies. The detection rate for norovirus RNA was improved. Further detection rate for genotype at initial RNA amount 10 ² copies the cleaving oligonucleotide by using two or more (E combination C) is improved, the initial RNA amount 10 in combination E is the most preferred combination ^The detection rate for genotype in ² copies improved to 5 out of 7 species.

From the above, in order to detect with high sensitivity against a wide range of genotypes of Norovirus, it is important to select a cleavage oligonucleotide, and it is preferable to use two or more cleavage oligonucleotides. .

The structure of the nucleic acid probe labeled with the intercalating fluorescent dye prepared in Example 2. B1, B2, B3 and B4 represent bases. According to the method of Ishiguro et al. (Non-Patent Document 4), an intercalating fluorescent dye (oxazole yellow) is bound to a phosphodiester moiety via a linker. In order to prevent an extension reaction from the hydroxyl group at the 3 'end, the hydroxyl group at the 3' end is modified with glycolic acid.

Claims (2)

A method for detecting Norovirus RNA in a sample, comprising: (1) using a specific nucleic acid sequence in Norovirus RNA as a template, overlapping a 5 ′ end site of a homologous region with the first primer in the specific base sequence, Cleaving the RNA at the 5 ′ terminal site of the specific base sequence with a cleavage oligonucleotide complementary to a region adjacent to the site in the 5 ′ direction and ribonuclease H (RNase H);
(2) The first base, the second primer, and the RNA-dependent DNA polymerase, RNase H, and DNA-dependent DNA polymerase with a promoter sequence added to the 5 ′ end, and the specific base downstream of the promoter sequence Generating a double-stranded DNA comprising the sequence;
(3) generating an RNA transcript comprising the specific base sequence by RNA polymerase using the double-stranded DNA as a template;
(4) 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;
(5) measuring the amount of the RNA transcript,
And the cleavage oligonucleotide comprises at least two kinds of oligonucleotides selected from oligonucleotides each having a sufficiently complementary sequence to SEQ ID NOs: 1 to 3, and the first primer is SEQ ID NO: 4 A method for detecting norovirus RNA, comprising a sufficiently homologous sequence, wherein the second primer comprises a sequence sufficiently complementary to SEQ ID NO: 5. In the method for detecting norovirus RNA, in the step (5) of measuring the amount of RNA transcript, the signal is present in the presence of a nucleic acid probe designed to change signal characteristics when a complementary double strand is formed with the target RNA. The detection method according to claim 1, wherein the detection method is performed by measuring a change, and the nucleic acid probe has a sequence sufficiently complementary to SEQ ID NO: 6.

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