JP2010004793A - Method for detecting norovirus rna and detection reagent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To detect norovirus RNA irrespective of gene group/genotype at a time. <P>SOLUTION: The amount of an amplified RNA product is measured by a nucleic acid probe designed so that the signal characteristics may be changed when the amplified RNA forms a complemental double-stranded chain in an RNA amplification step comprising a step of forming a double-stranded DNA by forming a double-stranded DNA containing a promoter sequence by using a combination of oligonucleotides comprising a first primer having higher hybridization efficiency to a homologous or complementary nucleic acid sequence in the norovirus RNA, and a second primer (the promoter sequence is added to the 5' terminus of one of the primers) by a reverse transcription enzyme, forming an RNA transcription product by using the double-stranded DNA as a template by an RNA polymerase, and forming the double-stranded DNA by consequently using the RNA transcription product as the template of the DNA synthesis by the reverse transcription enzyme. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for detecting norovirus RNA rapidly and with high sensitivity, and a detection reagent using the method. 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 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. In contrast, norovirus detection is currently 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 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, there is the method of Ishiguro et al. (See Patent Document 6 and Non-Patent Document 3). 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, 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 Japanese Patent Laid-Open No. 8-211050 JP 2001-13147 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) Infectious Disease Development Trend Survey Weekly Report, 6 (11), 14-19 (2004) Ishiguro T. et al, Nucleic Acids Res. , 24, 4992-4997 (1996)

  Noroviruses are roughly divided into two gene groups, genogroup I (GI) and genogroup II (GII), according to genotype. Further, for each gene group, GI is currently classified into genotypes 1 to 14 and GII is classified into genotypes 1 to 17 (see Non-Patent Document 4). 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, six sequences (Ciba (No. AB042808), Norwalk (No. NC_001959), Southampton (No. L07418), Chamberwell (No. AF145896), Hawaii (No. U07611), HuC (No. U07611), as sequences of Norovirus on GenBank. Even by examining the homology of No. AY032605)), there is no region of 20 bases or more having the same base sequence between genotypes.

  Against this background, the inventors have previously made a wide range of genotypes of norovirus RNA belonging to GI (hereinafter referred to as Norovirus GI RNA) or Norovirus RNA belonging to GII (hereinafter referred to as Norovirus GII RNA). On the other hand, a method of detecting rapidly and with high sensitivity has been found (Japanese Patent Application Nos. 2007-164921 and 2007-182904). However, in the above method, since the detection of Norovirus GI RNA and the detection of Norovirus GII RNA need to be performed separately, in order to determine whether Norovirus RNA is present in the sample, the same sample is used as a Norovirus GI RNA detection system. And norovirus GII RNA detection system. Since there is no difference in clinical symptoms due to the difference in the norovirus gene group / genotype, a method capable of detecting norovirus RNA at once regardless of the gene group or genotype is desired in the clinical field.

  As a result of intensive studies to solve the above problems, the present inventor has been able to detect Norovirus RNA quickly and with high sensitivity regardless of the gene group / genotype by a single measurement.

A first invention is a method for detecting Norovirus RNA in a sample, comprising:
(1) A first primer and a second primer in which a specific nucleotide 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, using a DNA-dependent DNA polymerase, generating a double-stranded DNA containing a promoter sequence and the specific base sequence downstream of the promoter sequence;
(2) a step of generating an RNA transcript comprising the specific base sequence or a complementary sequence thereof 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,
A mixture of oligonucleotides having a sequence sufficiently homologous to the sequence set forth in SEQ ID NO: 1 and oligonucleotides having a sequence sufficiently homologous to the sequence set forth in SEQ ID NO: 2 And the second primer is composed of an oligonucleotide having a sequence sufficiently complementary to the sequence shown in SEQ ID NO: 3, and a method for detecting norovirus RNA.

  According to a second invention, in the method for detecting Norovirus RNA, the first primer is selected from oligonucleotides having a sequence sufficiently homologous to at least one of the sequences set forth in SEQ ID NO: 4 or 5. A mixture of at least one oligonucleotide and an oligonucleotide having a sequence sufficiently homologous to the sequence set forth in SEQ ID NO: 6, wherein the second primer corresponds to the sequence set forth in SEQ ID NO: 7-9 The detection method according to the first invention, wherein the detection method comprises at least one oligonucleotide selected from oligonucleotides each having a sufficiently complementary sequence.

  According to a third invention, in the method for detecting Norovirus RNA, the first primer comprises a mixture of three kinds of oligonucleotides comprising the sequences of SEQ ID NOs: 4 to 6, and the second primer comprises SEQ ID NOs: 18 to 20. The detection method according to the second invention, comprising a mixture of three kinds of oligonucleotides having the described sequences.

According to a fourth aspect of the present invention, in the method for detecting Norovirus RNA, the region overlaps with the 5 ′ terminal site of the homologous region with the first primer in the specific nucleotide sequence and is 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 cleaving oligonucleotide and RNase H, the first primer, the second primer, and the RNA further added with a promoter sequence at the 5 ′ end Characterized in that it is carried out before the step of generating a double-stranded DNA comprising a promoter sequence and the specific base sequence downstream of the promoter sequence with a dependent DNA polymerase, RNase H, and DNA-dependent DNA polymerase;
The cleaving oligonucleotide is at least one oligonucleotide selected from oligonucleotides having a sequence sufficiently complementary to at least one of SEQ ID NOs: 10 to 12, and at least any of SEQ ID NOs: 13 to 15 The detection method according to any one of the first to third inventions, wherein the detection method comprises at least one oligonucleotide selected from oligonucleotides having a sequence sufficiently complementary to each other.

  A fifth invention is the nucleic acid designed so that the signal characteristic changes when the step (4) of measuring the amount of RNA transcript in the method for detecting norovirus RNA forms a complementary double strand with the target RNA. The nucleic acid probe is made by measuring the signal change in the presence of a probe, and the nucleic acid probe is an oligonucleotide having a sequence sufficiently complementary to SEQ ID NO: 16, and an oligonucleotide having a sequence sufficiently complementary to SEQ ID NO: 17. The detection method according to the fourth aspect of the invention, comprising nucleotides.

A sixth invention is a reagent for detecting Norovirus RNA in a sample,
(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 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 comprising the specific base sequence or a complementary sequence thereof 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,
A reagent for performing a detection method comprising: at least an oligonucleotide having a sequence sufficiently homologous to the sequence set forth in SEQ ID NO: 1 and a sequence set forth in SEQ ID NO: 2 Detection of norovirus RNA, characterized in that it comprises a mixture of oligonucleotides of sufficiently homologous sequence and wherein said second primer comprises an oligonucleotide of a sequence sufficiently complementary to the sequence set forth in SEQ ID NO: 3 It is a reagent.

  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 or its complementary sequence is amplified. Double-stranded DNA serving as a template for transcription of the RNA transcript has a specific base sequence in the sense strand or antisense strand downstream of the RNA polymerase promoter sequence. The 5 ′ end site of the homologous region with the first primer in the present invention consists of a partial sequence containing the 5 ′ end of the homologous region within the specific base sequence, and this site is a fragment of the cleavage oligonucleotide described later. It is a site where the homologous region between the complementary region and the first primer overlaps. 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 are preferred. The promoter sequence may be added with a transcription initiation region that is known to affect transcription efficiency.

  A sufficiently complementary sequence in the present invention is highly specific and highly efficient for a specific base sequence under optimized nucleic acid amplification reaction conditions (salt concentration, oligonucleotide concentration, reaction temperature, etc.). A sequence capable of hybridization. In addition, a sufficiently homologous sequence in the present invention refers to a sequence that can specifically and efficiently hybridize to a completely complementary sequence of a specific base sequence under optimized nucleic acid amplification reaction conditions. 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.

  As described above, there are currently 14 genotypes in Norovirus GI RNA and 17 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. (Japanese Patent Application No. 2007-164921). 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 (Japanese Patent Application No. 2007-182904).
However, as described above, the base sequence homology between Norovirus GI RNA and Norovirus GII RNA is as low as 40 to 50%. Therefore, in the combination of oligonucleotides optimized for Norovirus GI RNA and Norovirus GII RNA, It is difficult to detect norovirus RNA rapidly and with high sensitivity by one measurement regardless of the gene group / genotype.

  In addition, it is considered that a system for detecting Norovirus RNA at a time regardless of gene group / genotype can be constructed by simply mixing combinations of oligonucleotides optimized for Norovirus GI RNA and Norovirus GII RNA. When norovirus RNA is detected using the above combination, the number of primers increases, so that a nonspecific amplification product such as a primer dimer is likely to be formed, and as a result, there is a concern that the detection sensitivity to norovirus RNA decreases.

  Therefore, as a result of intensive studies, the present inventors have found a method for detecting norovirus RNA rapidly and with high sensitivity by a single measurement regardless of the gene group / genotype.

  That is, in the present invention, the first primer is an oligonucleotide having a sequence sufficiently homologous to the sequence described in SEQ ID NO: 1 homologous to a part of Norovirus GI RNA, and a SEQ ID NO: homologous to a part of Norovirus GII RNA. 2 consisting of an oligonucleotide having a sequence sufficiently homologous to the sequence described in 2, and the second primer being a sequence sufficiently complementary to the sequence described in SEQ ID NO: 3 that is homologous to a part of the Norovirus GI RNA Consisting of oligonucleotides.

  In the present invention, more preferably, the first primer is at least one selected from oligonucleotides having a sequence sufficiently homologous to the sequence set forth in SEQ ID NO: 4 or 5 that is homologous to a part of Norovirus GI RNA. An oligonucleotide, and an oligonucleotide having a sequence sufficiently homologous to the sequence set forth in SEQ ID NO: 6 that is homologous to a portion of Norovirus GII RNA, and wherein the second primer is a sequence that is homologous to a portion of Norovirus GI RNA It consists of at least one kind of oligonucleotide selected from oligonucleotides having a sequence sufficiently complementary to the sequences of Nos. 7 to 9, respectively.

  In a preferred embodiment of the present invention, the first primer consists of three oligonucleotides consisting of the sequences shown in SEQ ID NOs: 4 to 6, and the second primer consists of three kinds of sequences consisting of the sequences shown in SEQ ID NOs: 18 to 20. It consists of an oligonucleotide.

  Furthermore, as one aspect of the present invention, the Norovirus RNA is cleaved at the 5 'end site of a specific base sequence in the RNA before becoming a template for cDNA synthesis. By cleaving at the 5 ′ end site of the specific base sequence, after cDNA synthesis, a DNA strand complementary to the promoter sequence of the first primer hybridized to the cDNA is extended to 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). A method of cleaving an RNA portion of an 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, etc. . It is preferable to use a hydroxyl group at the 3 'end of the cleavage oligonucleotide that has been appropriately modified to prevent an extension reaction, for example, amination.

  In a preferred embodiment of the present invention, the cleavage oligonucleotide is at least one selected from sequences sufficiently complementary to the sequences set forth in SEQ ID NOs: 10 to 12 that are homologous to a part of Norovirus GI RNA. Use of a cleavage oligonucleotide and at least one kind of cleavage oligonucleotide selected from sequences sufficiently complementary to the sequences of SEQ ID NOs: 13 to 15 homologous to a part of Norovirus GII RNA it can.

  In the present invention, SEQ ID NOs: 4 and 10 are partial sequences of Southampton (GI / 2) (GenBank No. L07418), and SEQ ID NO: 5 is a partial sequence and sequence of Desert Shield (GI / 3) (GenBank No. U04469). Nos. 7 and 11 are partial sequences of Chiba (GI / 4) (GenBank No. AB042808), SEQ ID Nos. 8 and 12 are partial sequences of WUG1 (GI / 8) (GenBank No. AB087233), and SEQ ID No. 9 is Saitama T25G1 ( GI / 14) (GenBank No. AB112100), SEQ ID NOS: 6 and 13 are Lorsdale (GII / 4) (GenBank No. X86557) partial sequences, SEQ ID NO: 15 is Saitama U3 (GII / 6) GenBank No.AB039776) is a partial sequence of.

  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. In one embodiment of the present invention, the nucleic acid probe labeled with the intercalating fluorescent dye is an oligonucleotide having a sequence sufficiently complementary to SEQ ID NO: 16 homologous to a part of the Norovirus GI RNA, and the Norovirus GII RNA. An oligonucleotide with a sequence sufficiently complementary to SEQ ID NO: 17 that is partially homologous can be used.

  In one embodiment of the present invention, the cleavage oligonucleotide is at least one oligonucleotide selected from oligonucleotides having a sequence sufficiently complementary to SEQ ID NOs: 10 to 12 and SEQ ID NOs: 13 to 15, respectively. Each comprises at least one oligonucleotide selected from oligonucleotides of sufficiently complementary sequence. The first primer has a promoter sequence at its 5 ′ end, and is at least one oligo selected from oligonucleotides having sequences sufficiently homologous to the sequence shown in SEQ ID NO: 4 or 5. It consists of a nucleotide and an oligonucleotide of sequence sufficiently homologous to SEQ ID NO: 6. The second primer consists of an oligonucleotide having a sequence sufficiently complementary to SEQ ID NOs: 7 to 9, respectively. Furthermore, the intercalating fluorescent dye-labeled nucleic acid probe is preferably an oligonucleotide having a sequence sufficiently complementary to SEQ ID NO: 16 and an oligonucleotide having a sequence sufficiently complementary to SEQ ID NO: 17.

  More preferably, the cleavage oligonucleotide is an oligonucleotide consisting of the sequence shown in SEQ ID NO: 21 to 26, the first primer is an oligonucleotide consisting of the sequence shown in SEQ ID NO: 4 to 6, and the second primer is An oligonucleotide consisting of the sequence shown in SEQ ID NOs: 18 to 20, and the intercalator fluorescent dye-labeled nucleic acid probe consisting of an oligonucleotide consisting of the sequences shown in SEQ ID NOs: 27 and 28.

  In the method for detecting norovirus RNA of 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. An enzyme having RNA polymerase activity in addition to a reverse transcriptase having 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 It is also possible to supplement by adding an enzyme having RNase H activity as necessary. The reverse transcriptase is preferably AMV reverse transcriptase, MMLV reverse transcriptase, HIV reverse transcriptase, or derivatives thereof widely used in molecular biological experiments, and most preferably AMV reverse transcriptase and derivatives thereof. Further, as the enzyme having RNA polymerase activity, bacteriophage-derived T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and derivatives thereof widely used in molecular biological experiments and the like can be used.

  In one embodiment of the present invention, the cleaving oligonucleotide is added to Norovirus RNA in a 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 degraded by the RNase H activity of the reverse transcriptase and dissociated, whereby the first primer is bound 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, and particularly preferably set in the range of 40 to 50 ° 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 a complementary double strand is formed with a nucleic acid probe labeled with an intercalating fluorescent dye and the target nucleic acid, the intercalating fluorescent dye portion intercalates with the complementary double strand portion. In the presence of a nucleic acid probe designed so that the fluorescence characteristics are changed by the above-mentioned method, the nucleic acid amplification step is carried out to measure the change in fluorescence characteristics (see Patent Document 6 and Non-Patent Document 3).

  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 target RNA on the RNA transcript, via an appropriate linker at the end, phosphodiester part or base part. It has a structure in which an intercalating fluorescent 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 (Patent Document 7 and Non-Patent Document 5). reference).

  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 8 and Non-Patent Document 5). . 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, 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: 40) to the 5 ′ end of the sequence described in SEQ ID NO: 4), A sequence in which a T7 promoter sequence (SEQ ID NO: 40) is added to the 5 ′ end of the sequence described in SEQ ID NO: 5 and a sequence in which a T7 promoter sequence (SEQ ID NO: 40) is added to the 5 ′ end of the sequence described in SEQ ID NO: 6 ), A second primer (sequences shown in SEQ ID NOs: 18 to 20), a nucleic acid probe labeled with an intercalating fluorescent dye (sequences shown in SEQ ID NOs: 27 and 28), a cleavage oligonucleotide (SEQ ID NOs: 21 to 26) Sequence shown), AMV reverse transcriptase, T7 RNA polymerase, buffer, magnesium salt, potassium salt, nucleoside-triphosphate An amplification reagent containing ribonucleoside-triphosphate and dimethyl sulfoxide (DMSO) is added and reacted at a constant temperature of 35 to 65 ° C. (preferably 40 to 50 ° C.) and simultaneously the fluorescence intensity of the reaction solution is changed over time. Provide a way to measure.

  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 30 minutes. 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 an operation of dispensing a certain amount of sample into such a single container is performed, 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, norovirus RNA can be measured simultaneously with high specificity, high sensitivity, rapidity, simplicity, constant temperature, and one step regardless of gene group / genotype.

The present invention
(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, using a DNA-dependent DNA polymerase, generating a double-stranded DNA containing a promoter sequence and the specific base sequence downstream of the promoter sequence;
(2) a step of generating an RNA transcript comprising the specific base sequence or a complementary sequence thereof 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,
In the method for detecting Norovirus RNA, an oligonucleotide having a sequence sufficiently homologous to the sequence set forth in SEQ ID NO: 1 wherein the first primer is homologous to a portion of Norovirus GI RNA, and a portion of Norovirus GII RNA The second primer is sufficiently homologous to the sequence set forth in SEQ ID NO: 3 that is homologous to a portion of the Norovirus GI RNA. It is characterized by comprising an oligonucleotide having a complementary sequence. According to the present invention, it is possible to rapidly amplify and detect a small amount of Norovirus RNA contained in a sample regardless of the gene group / genotype at a constant temperature.

  Further, in the step (4) of measuring the amount of RNA transcript, the amplified norovirus RNA is identified by coexisting with a nucleic acid probe designed to change the signal characteristics when a complementary double strand is formed with the target RNA. By measuring the increase in fluorescence intensity derived from nucleic acid over time, the detection of norovirus RNA can be performed simply, rapidly and with high sensitivity.

  The detection reagent for Norovirus RNA using the detection method of the present invention can detect Norovirus RNA quickly and with high sensitivity regardless of the gene group / genotype in a single measurement. Therefore, it is possible to quickly and easily determine whether or not norovirus is present in clinical specimens such as feces and vomit in clinical settings, and is useful for the prevention of subsequent norovirus infection and investigation of norovirus infection routes. .

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by these Examples.

Example 1 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) is 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.

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 ＧＩ ＲＮＡの遺伝子型全１４種のうちの８種、ノロウイルス ＧＩＩ ＲＮＡの遺伝子型全１７種のうちの７種と、それぞれ一部ではあるが、
（Ａ）ＧＩ／１、ＧＩ／６、およびＧＩ／８との間
（Ｂ）ＧＩ／４、ＧＩ／５、およびＧＩ／９との間
（Ｃ）ＧＩ／３、ＧＩ／１０、ＧＩ／１１、ＧＩ／１２、ＧＩ／１３、およびＧＩ／１４との間
（Ｄ）ＧＩＩ／７、ＧＩＩ／８、ＧＩＩ／９およびＧＩＩ／１３との間
（Ｅ）ＧＩＩ／１１、ＧＩＩ／１４、およびＧＩＩ／１６との間
（Ｆ）ＧＩＩ／１、ＧＩＩ／１２、およびＧＩＩ／１５との間
（Ｇ）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４）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ＲＮＡを検出可能であることが予想される。

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 standard genotypes for which standard RNA was prepared this time are 8 types of all 14 types of norovirus GI RNA and 7 types of all 17 types of norovirus GII RNA genotype,
(A) Between GI / 1, GI / 6, and GI / 8 (B) Between GI / 4, GI / 5, and GI / 9 (C) GI / 3, GI / 10, GI / 11 , GI / 12, GI / 13, and GI / 14 (D) Between GII / 7, GII / 8, GII / 9, and GII / 13 (E) GII / 11, GII / 14, and GII (F) GII / 1, GII / 12, and GII / 15 (G) GII / 2, GII / 5, and GII / 10 each have high homology (Non-patent From the literature 4), if all the standard RNAs of the genotype prepared this time can be detected, it is expected that norovirus RNAs belonging to all genotypes other than the special genotype GII / 17 can be detected.

Example 2 Preparation of nucleic acid probe labeled with intercalating fluorescent dye A nucleic acid probe labeled with an intercalating fluorescent dye was prepared. According to the method described in Non-Patent Document 5, between the 12th C and 13th A from the 5 ′ end of the sequence described in SEQ ID NO: 27, the 12th C from the 5 ′ end of the sequence described in SEQ ID NO: 28 Oxazole yellow was bound to the phosphodiester moiety between the 12th C and the 13th A from the 5 ′ end of the sequence shown in SEQ ID NO: 36 between the A and the 13th A through a linker, respectively. An oxazole yellow labeled nucleic acid probe was prepared (FIG. 1).

Example 3 Detection of Norovirus RNA Using 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 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, the INAF probe, and the cleaving oligonucleotide described in the combinations A and B are the Norovirus GI RNA (combination combination) previously discovered by the inventors. A, a combination of oligonucleotides that detects a wide range of genotypes of Norovirus GII RNA (Combination B, Japanese Patent Application No. 2007-182904) quickly and with high sensitivity. The combination C is a system in which the oligonucleotide used in the combination A and the oligonucleotide used in the combination B are simply mixed. Combinations D to F are the system of the present invention. Of the oligonucleotides described in combinations D to F, SEQ ID NOs: 4 and 5 are included in SEQ ID NO: 1, SEQ ID NO: 6 is included in SEQ ID NO: 2, and SEQ ID NOs: 18 to 20 are complementary strands of SEQ ID NO: 3. include.

（１）前記標準ＲＮＡをＲＮＡ希釈液（１０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ８．０）、１ｍＭ ＥＤＴＡ、０．２５Ｕ／μＬ リボヌクレアーゼインヒビター、５．０ｍＭ ＤＴＴ）を用いて、ＧＩ 標準ＲＮＡは３００コピー／５μＬまたは１０^３コピー／５μＬに、ＧＩＩ 標準ＲＮＡは１０^２コピー／５μＬまたは１０^３コピー／５μＬに、それぞれなるよう希釈し、これらをＲＮＡ試料として用いた。
（２）以下の組成の反応液２０μＬを０．５ｍＬ容量ＰＣＲ用チューブ（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) The standard RNA was diluted with an RNA diluent (10 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA, 0.25 U / μL ribonuclease inhibitor, 5.0 mM DTT), and GI standard RNA was 300 copies / to 5 [mu] L or 10 ³ copies / 5 [mu] L, the GII standard RNA are 10 ² copies / 5 [mu] L or 10 ³ copies / 5 [mu] L, it was diluted to a respectively, using these as the RNA sample.
(2) 20 μL of the reaction solution having the following composition was dispensed into a 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and 5 μL of the RNA sample was added thereto.

Composition of reaction solution: concentration is final concentration after addition of enzyme solution (in 30 μL) 60 mM Tris-HCl buffer (pH 8.6)
18 mM Magnesium chloride 100 mM Potassium chloride 1 mM DTT
0.25 mM dATP, dCTP, dGTP, dTTP each
Each 3 mM ATP, CTP, UTP, GTP
3.6 mM ITP
Each 0.75 μM first primer: each primer has a T7 promoter sequence (SEQ ID NO: 40) added to the 5 ′ end of the base sequence described in each SEQ ID NO. Each 1 μM second primer 20 nM INAF probe (combination C To F, 10 nM each): The probe prepared in Example 2 0.16 μM each Oligonucleotide for cleavage: The hydroxyl group at the 3 ′ end of the oligonucleotide is modified with an amino group 6U ribonuclease inhibitor (Takara Bio) Made)
13% DMSO
(3) After the above reaction solution was kept at 43 ° C. for 5 minutes, 5 μL of an enzyme solution having the following composition and kept at 43 ° C. for 2 minutes in advance was added.

Composition of enzyme solution: final concentration during reaction (in 30 μL) 2.0% sorbitol 6.4 U 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 at the same time the fluorescence intensity of the reaction solution (excitation wavelength: 470 nm, fluorescence wavelength: 520 nm) Measurement was performed for 30 minutes over time.

The time when the enzyme is added is defined as positive when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value of the predetermined time by the fluorescence intensity ratio of the background) exceeds 1.2. Tables 3 and 4 show the results of the detection time. Table 3 shows the results when 10 ³ copies of GI and / or GII standard RNA were measured using the oligonucleotide combinations shown in combinations A to D in Table 2, and Table 4 shows combinations D in Table 2. the results measured with 300 copies of the GI standard RNA and 10 ² copies of GII standard RNA using combinations of oligonucleotides shown in F from, respectively. In Tables 3 and 4, “−” indicates a sample that has not been measured, N.P. D. Means a sample having a fluorescence intensity ratio of less than 1.2 (negative determination) 30 minutes after the enzyme was added.

以前発明者らの検討で、１０^３コピーのＧＩまたはＧＩＩ 標準ＲＮＡを遺伝子型を問わず、３０分以内に検出する系を見出している（特願２００７−１６４９２１号、特願２００７−１８２９０４号）。そこで、当該検出系（組み合わせＡおよびＢ）で用いたオリゴヌクレオチドを単純に混合した系（組み合わせＣ）で１０^３コピーの標準ＲＮＡを測定した。その結果、表３に示すように、ＧＩ 標準ＲＮＡを測定したときは、組み合わせＡを用いた系では測定した８遺伝子型全てを３０分以内に検出した一方、組み合わせＣを用いた系では検出した遺伝子型が２つに激減した。また、ＧＩＩ 標準ＲＮＡを測定したときは、組み合わせＢを用いた系、組み合わせＣを用いた系、ともに、測定した８遺伝子型全てを３０分以内に検出していたものの、検出時間は全ての遺伝子型で組み合わせＣを用いた系の方が遅れていた。試料中のＲＮＡ量が等しい場合、検出時間が早いほど増幅効率が高いため、組み合わせＣを用いた系は、組み合わせＢを用いた系と比較し、増幅効率が低下しているといえる。よって、ノロウイルス ＧＩまたはＧＩＩ 標準ＲＮＡの広範囲な遺伝子型に対して迅速、高感度に検出する系が見出されたとしても、それらの系で使用しているオリゴヌクレオチドを単純に混合するだけでは、一度の測定でノロウイルスＲＮＡを遺伝子群／遺伝子型を問わず検出する系を構築するのは困難であることがわかる。

Previously, the inventors have found a system that detects 10 ³ copies of GI or GII standard RNA within 30 minutes regardless of genotype (Japanese Patent Application Nos. 2007-164921 and 2007-182904). . Therefore, 10 ³ copies of standard RNA were measured in a system (combination C) in which the oligonucleotides used in the detection system (combination A and B) were simply mixed. As a result, as shown in Table 3, when the GI standard RNA was measured, all the 8 genotypes measured in the system using the combination A were detected within 30 minutes, whereas the system using the combination C was detected. The genotype was drastically reduced to two. In addition, when GII standard RNA was measured, both the system using combination B and the system using combination C detected all 8 genotypes measured within 30 minutes, but the detection time was all genes. The system using combination C in the mold was delayed. When the amount of RNA in the sample is equal, the amplification efficiency is higher as the detection time is earlier. Therefore, it can be said that the amplification efficiency is lower in the system using the combination C than in the system using the combination B. Therefore, even if a system that detects rapidly and sensitively for a wide range of genotypes of Norovirus GI or GII standard RNA is found, simply mixing the oligonucleotides used in those systems It can be seen that it is difficult to construct a system that detects Norovirus RNA regardless of gene group / genotype by a single measurement.

Receiving said result, in the newly system of the present invention that non-specific amplification such primer dimers were obtained conducted intensive study combinations unlikely oligonucleotides (combination D), the 10 ³ copies of standard RNA When measured, all the measured standard RNAs (GI 8 genotype, GII 7 genotype) were detected within 20 minutes.

Further, in the system of the present invention (F combinations D), 300 copies of GI standard RNA, and 10 ² copies of GII standard RNA were measured. As a result, as shown in Table 4, RNA amount used even though less than in Table 3 (10 ³ copies), both the system of the present invention, compared with a system using a combination C The number of genotypes detected (total of GI and GII) increased (combination C: 9 genotype, combination D: 15 genotype, combination E: 13 genotype, combination F: 12 genotype). Among them, the system using the combination D is the most preferable system because it detects all the measured standard RNAs (GI 8 genotype, GII 7 genotype) within 30 minutes.

From the above, by using the first primer, the second primer, the intercalating fluorescent dye-labeled nucleic acid probe, and the cleaving oligonucleotide described in the present invention, the norovirus RNA can be determined regardless of the gene group / genotype in one measurement. It was shown that detection was possible quickly and with high sensitivity.

The structure of the intercalating fluorescent dye-labeled nucleic acid probe produced in Example 2. B1, B2, B3 and B4 represent bases. A probe in which an intercalating fluorescent dye (oxazole yellow) is bound to a phosphodiester moiety via a linker according to the method of Ishiguro et al. (Non-patent Document 5). 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 (6)

A method for detecting Norovirus RNA in a sample comprising:
(1) A first primer and a second primer in which a specific nucleotide 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, using a DNA-dependent DNA polymerase, generating a double-stranded DNA containing a promoter sequence and the specific base sequence downstream of the promoter sequence;
(2) a step of generating an RNA transcript comprising the specific base sequence or a complementary sequence thereof 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,
A mixture of oligonucleotides having a sequence sufficiently homologous to the sequence set forth in SEQ ID NO: 1 and oligonucleotides having a sequence sufficiently homologous to the sequence set forth in SEQ ID NO: 2 And the second primer comprises an oligonucleotide having a sequence sufficiently complementary to the sequence shown in SEQ ID NO: 3. In the method for detecting norovirus RNA, the first primer is at least one oligonucleotide selected from oligonucleotides having a sequence sufficiently homologous to at least one of the sequences shown in SEQ ID NO: 4 or 5. And a mixture of oligonucleotides of sequences sufficiently homologous to the sequence set forth in SEQ ID NO: 6, wherein the second primer is sufficiently complementary to each of the sequences set forth in SEQ ID NOs: 7-9 It consists of at least 1 type of oligonucleotide chosen from the oligonucleotide of the arrangement | sequence, The detection method of Claim 1 characterized by the above-mentioned. In the method for detecting norovirus RNA, the first primer is composed of a mixture of three kinds of oligonucleotides consisting of the sequences shown in SEQ ID NOs: 4 to 6, and the second primer is composed of three kinds consisting of the sequences shown in SEQ ID NOs: 18 to 20 The detection method according to claim 2, comprising a mixture of oligonucleotides. In the method for detecting norovirus RNA, a cleavage oligonucleotide complementary to a region adjacent to the 5 ′ end of the homologous region with the first primer in the specific base sequence overlapping with the 5 ′ end region, Cleaving the RNA at the 5 ′ end of the specific base sequence with RNase H, the first primer having a promoter sequence added to the 5 ′ end, the second primer, an RNA-dependent DNA polymerase, and RNase H and a DNA-dependent DNA polymerase are performed before the step of generating a double-stranded DNA containing a promoter sequence and the specific base sequence downstream of the promoter sequence,
The cleaving oligonucleotide is at least one oligonucleotide selected from oligonucleotides having a sequence sufficiently complementary to at least one of SEQ ID NOs: 10 to 12, and at least any of SEQ ID NOs: 13 to 15 The detection method according to any one of claims 1 to 3, wherein the detection method comprises at least one kind of oligonucleotide selected from oligonucleotides having a sequence sufficiently complementary to each other. In the method for detecting norovirus RNA, the step (4) of measuring the amount of RNA transcript is carried out in the presence of a nucleic acid probe designed to change signal characteristics when a complementary double strand is formed with the target RNA. Wherein the nucleic acid probe is composed of an oligonucleotide having a sequence sufficiently complementary to SEQ ID NO: 16 and an oligonucleotide having a sequence sufficiently complementary to SEQ ID NO: 17 The detection method according to claim 4. A reagent for detecting norovirus RNA in a sample,
(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 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 comprising the specific base sequence or a complementary sequence thereof 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,
A reagent for performing a detection method comprising: at least an oligonucleotide having a sequence sufficiently homologous to the sequence set forth in SEQ ID NO: 1 and a sequence set forth in SEQ ID NO: 2 Detection of norovirus RNA, characterized in that it comprises a mixture of oligonucleotides of sufficiently homologous sequence and wherein said second primer comprises an oligonucleotide of a sequence sufficiently complementary to the sequence set forth in SEQ ID NO: 3 reagent.

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