JP4898210B2 - How to detect sapovirus - Google Patents

Description

  The present invention relates to a virus detection method.

  An electron micrograph of fecal material from a patient with mass gastroenteritis that occurred in the United States in 1972 discovered a small spherical virus with a surface structure of around 30 nm in diameter. Later, it was tentatively called SRSV (small spheroid virus) for viruses with such a shape by electron microscopy. This virus group included Norwalk virus, Sapporo virus, and astrovirus, but Norwalk virus and Sapporo virus belonged to Caliciviridae at the 8th International Virus Naming Committee held in August 2002. The former was named Norovirus and the latter was named Sapovirus. Norovirus has a diameter of 27 to 32 nm, and the genogroup is divided into two, and more than 30 genotypes exist.

  Four genotypes of sapovirus have been reported so far. Both viruses have a capsid as a surface structure and have a single-stranded RNA (7.5 Kb).

  According to the Infectious Disease Development Trend Survey Weekly Report (2004) reported by the Ministry of Health, Labor and Welfare (2004), norovirus was the most common pathogen detection information in winter, especially 241 cases of norovirus GII infection were detected in 19 prefectures, and GI 11 cases were detected, 39 G unknowns were detected, and a total of 291 cases were detected. In addition, 16 cases of sapoviruses and 20 cases of rotaviruses have been isolated and reported. Although it is estimated that sapovirus is less frequent than norovirus, it is listed as a causative virus for infant diarrhea.

  Detection of diarrhea virus from feces has already been carried out in many health laboratories and inspection centers targeting norovirus.

For the detection of norovirus, many mass processing methods such as PCR, real-time PCR, and TRC have been reported.
Arch Virol. 146.2115-2132 (2001) J.Med.Viro.65.413-417 (2001) Arch Virol. 147.1445-1451 (2002)

  There is a need for the development of a simple measurement system capable of mass processing of sapovirus, a kind of etiological virus that causes food poisoning and diarrhea in winter.

  Currently, several detection methods based on the RT-PCR method have been reported for sapovirus (Non-Patent Documents 1 to 3 above). However, in these conventional methods for detecting sapovirus, it is necessary to amplify a long gene region mainly to determine the DNA sequence and classify the genotype, and to confirm the detection, electrophoresis is also required. Therefore, the time and labor cost for detection are problems. Moreover, although detection by an electron microscope is also implemented, the problem that it is difficult to cope with a large amount of processing due to limited facilities is pointed out.

  In addition, sapoviruses are roughly classified into four genotypes, but the homology of the gene sequences is low and it was difficult to detect all genotypes at once with high sensitivity.

  The problem to be solved by the present invention is to provide a means for detecting a sapovirus present in feces and the like with high sensitivity and efficiency.

As a means for solving the above problems, the present inventor has found that a part of cDNA (part of RdRp region) using sapovirus gene RNA as a template has a high degree of conservation between the strains. The present invention has been completed by conceiving that a nucleic acid having a base sequence corresponding to the base sequence is used as an index.
That is, the present invention is a nucleotide sequence represented by SEQ ID NO: 5 as a cDNA of a sapovirus prototype (reference strain),
(1) In a base sequence corresponding to Nos. 5071 to 5094, a set of three or more + -strand nucleic acids (a-1) in which 15 to 24 bases are continuous is used as one gene amplification primer, and 5154 to 5178 Using a pair (b-2) of two or more negative-strand nucleic acids in which 15 to 25 bases are continuous in the base sequence corresponding to the number as the other gene amplification primer, or
(2) A group of three or more negative-strand nucleic acids (a-2) in which 15 to 24 bases are continuous in the base sequence corresponding to the numbers 5071 to 5094 is used as one gene amplification primer, and the numbers 5154 to 5178 Using a pair (b-1) of two or more + strand nucleic acids in which 15 to 25 bases are continuous in the base sequence corresponding to the above, as the other gene amplification primer,
A method of applying a gene amplification means to a gene obtained from a sample, and detecting a sapovirus in the sample using a gene detection probe obtained from the gene amplification product ,
(3) The above-mentioned three or more types of + strand nucleic acid sets (a-1) or − strand nucleic acid sets (a-2) are the same as the nucleic acids of SEQ ID NOS: 17, 18 and 19, respectively. Always included as a chain,
(4) The above-described two or more types of nucleic acid pairs (b-1) or -strand nucleic acid pairs (b-2) are the same as the nucleic acid pairs of SEQ ID NOS: 20 and 21, respectively. Always include as
(5) The nucleic acid probe for gene detection is a fluorescently labeled nucleic acid probe for gene detection including a + strand or − strand base sequence continuous with at least 10 bases among base sequences corresponding to 5101 to 5121 ,
The present invention provides a virus detection method (hereinafter also referred to as this detection method).

  The reference strain in the present invention is a sapovirus strain having a base sequence registered in GeneBank under Accession No. X86560. Further, “corresponding” means a base sequence corresponding to the gene region indicated by the base sequence of the cDNA of the reference strain as a result of gene analysis in a sapovirus containing each mutant strain. Specifically, it refers to a base sequence that can hybridize under stringent conditions (56 to 68 ° C. in the presence of 50 mM or more sodium ions) against the diffusion of the base sequence of the cDNA of the reference strain. means. More specifically, the base sequences corresponding to the gene regions indicated by the base sequences of the cDNAs of the above-mentioned reference strains of various sapoviruses shown in FIG. 1 are exemplified. Furthermore, as described above, “complementary” means that hybridization is possible under conditions that are stringent for diffusion of a certain base sequence (56 to 68 ° C., in the presence of 50 mM or more sodium ion). It means a base sequence having complementarity, and “complementary both base sequences” means a complementary base sequence having a −strand relationship to the + strand and / or a complement having a + strand relationship to the − strand. This means a base sequence including both of the target base sequences.

  The present invention provides a means for detecting a sapovirus present in stool or the like with high sensitivity and efficiency.

<Primers and probes for detection>
Currently, the Sapovirus strains registered in GeneBank are AJ251991, U65427, U95643, X86559, X86560, AF294739, U73124, U95644, AJ249939, AJ271056, U95645, AF435814, AY237420. Strains, AY603425 strain, NC_006554 strain, NC_010624 strain. These disclosed nucleotide sequences are cDNAs prepared using the gene RNA of each sapovirus strain as a template. The positional relationship between the base sequences of the genes of these sapovirus strains and the primer probes used in the examples of the present invention is shown in FIG. In FIG. 1, the name of the sapovirus strain is shown at the left end, and the uppermost row shows the base sequence of the AJ251991 strain. In the display of the base sequence of each strain, “.” Indicates that it is the same base as the AJ251991 strain, and when “−” indicates that the base does not exist. Regardless of the display in FIG. 1, the reference strain of the present invention is the X86560 strain, and the reference sequence number in the present invention is the sequence number of the X86560 strain in FIG. However, the base sequence number of each sapovirus strain may be used as a notation as necessary.

  The gene region of cDNA serving as an index for this detection method is a part of the RdRp region of sapovirus. The gene region shown in FIG. 1 is AJ251991 strain No. 665-886 (SEQ ID NO: 1 in the sequence listing) The base sequence numbers are counted from 1 (the same applies hereinafter), the U65427 strain is 695-916 (SEQ ID NO: 2), the U95643 strain is 743-964 (SEQ ID NO: 3), and the X86559 strain is 493-714. No. (SEQ ID NO: 4), X86560 strain (reference strain) is 5034-5255 (SEQ ID NO: 5), AF294739 strain is 720-947 (SEQ ID NO: 6), U73124 strain is 657-884 (SEQ ID NO: 7), The U95644 strain is 657-884 (SEQ ID NO: 8), the AJ249939 strain is 5038-5262 (SEQ ID NO: 9), the AJ271056 strain is 642-866 (SEQ ID NO: 10), and the U95645 strain is 743-964 (SEQ ID NO: 11). ) AF435814 stock is 658 ~ No. 867 (SEQ ID NO: 12), AY237420 strain is 5038-5262 (SEQ ID NO: 13), AY603425 strain is 5037-5264 (SEQ ID NO: 14), NC_006554 strain is 5037-5264 (SEQ ID NO: 15), NC_010624 strain is No. 5038 to 5262 (SEQ ID NO: 16).

  Specifically, when using knowledge about the conserved region of the sapovirus as an indicator for detecting the sapovirus, the conserved region is amplified using a nucleic acid amplification method, and the gene amplification product of the conserved region is amplified. It is assumed that As the nucleic acid amplification method, various nucleic acid amplification methods typically using the PCR method, for example, RT-PCR method, real-time PCR method, NASBA (Nucleic acid Sequence based Amplication) method, SDA (Strand Displacement Amplification) method, TRC ( A Transcription Reverse Transcription Concerted Reaction (Tosoh Corporation) method, a LAMP (Loop-Mediated Isothermal Amplification) method (Eiken Chemical Co., Ltd.), or the like can be used. Among these methods, it is preferable to use a real-time PCR method that allows easy quantification. Specific examples of the real-time PCR method include an intercalator method, a TaqMan probe method, a molecular beacon probe method, a cycling probe method, and the like, and any method can be used.

  In order to use these gene amplification methods, it is necessary to design and prepare a gene amplification method based on the selected nucleic acid amplification method. The gene amplification primer should sandwich the gene region intended for amplification with a + -primer that amplifies the gene in the forward direction of 5 ′ → 3 ′ and the reverse direction. It is usual to design. The number of bases in such a gene amplification primer must be at least correctly bound to the region to be complementarily bound in the template gene, and is complementary to the characteristic base sequence. It is necessary to ensure a minimum number of bases. The number of bases in such a gene amplification primer is at least 10 bases, preferably about 15 to 25 bases. In addition, it is preferable to design a gene amplification primer so as to have as close complementarity as possible to a region corresponding to 5 bases counted from the 3 'side of the region to be complementarily bound.

  In addition, it is preferable that the gene region to be the base of the gene amplification primer includes at least a complementary double nucleotide sequence corresponding to the conserved region in the gene amplification product obtained using the primer.

  As described above, the conserved region of the sapovirus gene that serves as an index in this detection method corresponds to the nucleotide sequence Nos. 5071 to 5178 (included in SEQ ID NO: 5) of the prototype (reference strain: X86560 strain) cDNA. It is the whole or a part of both complementary base sequences. Next, among the base sequence regions shown in SEQ ID NOs: 1 to 4 and 6 to 16, the base sequence corresponding to the base sequence of the conserved region of the reference strain (X86560 strain) is SEQ ID NO: 1 (AJ251991 strain) Gene) is 702 to 809, SEQ ID NO: 2 (U65427 strain gene) is 732 to 839, SEQ ID NO: 3 (U95643 strain) is 780 to 887, and SEQ ID NO: 4 (X86559 strain) is 530. No. 637, SEQ ID NO: 6 (AF294739 strain) is 757-864, SEQ ID NO: 7 (U73124 strain) is 694-801, SEQ ID NO: 8 (U95644 strain) is 694-801 SEQ ID NO: 9 (AJ249939 strain) is 5075-5158, SEQ ID NO: 10 (AJ271056 strain) is 679-786, SEQ ID NO: 11 (U95645 strain) is 780-887, SEQ ID NO: 12 ( AF435814) No. 02, 5075 to 5182 in SEQ ID NO: 13 (AY237420 strain), 5074 to 5181 in SEQ ID NO: 14 (AY603425 strain), 5074 to 5181 in SEQ ID NO: 15 (NC_006554 strain), In SEQ ID NO: 16 (NC_010624 strain), it is 5195-5182.

  As a part of the complementary base sequences corresponding to the nucleotide numbers 5071 to 5178 of the cDNA of the prototype (reference strain: X86560 strain), in particular, (1) the nucleotide sequence corresponding to the numbers 5071 to 5094, (2) 5101 A base sequence corresponding to -5121, and (3) a highly conserved region between sapovirus strains of 3 regions of the base sequence corresponding to 5154-5178, and this part is an indicator of detection (typically a probe) or It is preferable to use it as an essential part of a gene amplification primer. That is, the present detection method is such that the base sequence of the cDNA of the sapovirus prototype (reference strain) in the sample is (1) 5071 to 5094 in 10 bases or more and (3) 5154 to 5178 in 10 bases or more. It is preferable to adopt a mode in which a sapovirus is detected using a nucleic acid having complementary base sequences corresponding to one or more selected from the group consisting of continuous base sequences. These base sequences that are bases, (2) base sequences that are 10 bases or more continuous in Nos. 5101 to 5121, and sequence regions (1) to (3) are respectively enclosed by boxes in FIG.

( A ) When a gene in a sample is amplified using a primer for gene amplification whose essential part is the set of base sequence regions (1) and (2), the base sequence region (1) or It is preferable to use a probe whose essential part is (2).

( B ) When a gene in a sample is amplified using a gene amplification primer having the essential part of the pair of base sequence regions (2) and (3), the base sequence region (2 ) Or (3) is preferably used.

( C ) Further, when a gene in a specimen is amplified using a gene amplification primer having the essential part of the pair of base sequence regions (1) and (3), the base sequence region (1 ), (2) or (3) can be used as the essential part, but it is preferable to use a probe whose part is the region (2).

Thus, both base sequences corresponding to the base sequence regions (1) to (3) can be used as detection indicators in the present detection method or as essential parts of gene amplification primers, respectively. It is preferable to carry out the present detection method using a combination of the above ( C ). In particular, the base sequence region (2) is used as an essential part of the detection probe, and the base sequence regions (1) and (3) Is most preferably used as an essential part of the primer for gene amplification.

<Specific detection mode>
Samples to which this detection method is applied are all those for which sapovirus is to be detected. For example, when a sapovirus is to be detected in a food poisoning patient, typically, the feces of the food poisoning patient, and in some cases, vomit, blood, etc. can be used as a specimen. In addition, it is possible to use foods, deposits in food production facilities, clothes of food producers and food poisoning patients, and the like as specimens. Furthermore, it is also possible to use various sewage, drainage, seawater, river water, lake water, and other water-based water as a specimen. As a method for preparing genes from these specimens, an appropriate method can be selected according to the type of specimen to be used. In general, the specimen to be used is obtained by immersing or suspending the specimen in water or the like. From the obtained upper layer fraction, viral RNA can be extracted by a conventional method such as the acid phenol method (ACPC method: Acid Guanidin Phenol Chroloform method). Such a viral RNA is treated by a gene amplification method to be selected, for example, a cDNA complementary to the viral RNA using reverse transcriptase is prepared, and the nucleic acid sample is prepared using the above gene amplification primers. By performing gene amplification, a desired gene amplification product can be obtained (for confirmation of gene amplification product, for example, whether a gene amplification product of a desired size is recognized by electrophoresis, or This is performed using as an index whether or not a gene amplification product containing a specific sequence detected by a probe of a specific sequence is recognized). Further, by using the real-time PCR method, it is possible to quantitatively detect a desired gene amplification product while omitting the labor of electrophoresis.

  As described above, the detection nucleic acid probe for detecting a specific gene amplification product obtained by the gene amplification method is essentially any one of the base sequence regions (1) to (3), particularly preferably (2). A probe having a nucleic acid The nucleic acid probe is, for example, a nucleic acid labeled with a fluorescent label, an isotope label, a dye label, etc., and using these labels, the presence of a base sequence to be detected in a gene amplification product is determined by hybridization, etc. The method of confirming negative or positive by can be used. Generally, these detection means are performed after the completion of the gene amplification operation. In this method, after the gene amplification operation reaction, the tube must be opened and the analysis sample must be taken out. In addition to increasing the chances of contaminating equipment and reagents with gene amplification products, time and labor are required. Could increase the chances that Therefore, in order to avoid dangers such as contamination, and to reduce the time spent for detection operations as much as possible, it is possible to monitor specific base sequences in gene amplification products in real time during the process of gene amplification operations. It is preferable to perform a real-time PCR method. The real-time PCR method is a method for monitoring and analyzing the amount of amplification of a gene amplification product by a PCR reaction in real time. Specifically, a PCR reaction is performed using a known amount of DNA diluted serially as a standard, and based on this, in the region where gene amplification occurs exponentially, the number of cycles with a constant amount of amplified product and the initial In this method, a calibration curve obtained from the correlation with the amount of DNA is prepared, and the target amount of DNA in the sample is measured based on the calibration curve. As described above, examples of the real-time PCR method include an intercalator method, a TaqMan probe method, a molecular beacon probe method, and a cycling probe method.

  The intercalator method is a method in which a reagent that emits fluorescence by binding to double-stranded DNA (intercalator) is added to the PCR reaction system, and the amount of gene amplification product generated is monitored by detecting the fluorescence. Is possible.

Molecular beacon probes method, production of the gene amplification product during the amplification process (after the amplification process is possible) can be used to monitor a fluorescent hairpin hybridization probes [Molecular beacon probes (Molecular beacon The Probe)] (Nature Biotechnology 1998 16: 49-53). The ends of the nucleic acid constituting the molecular beacon probe are complementary to each other. Usually, these ends are bonded to each other to form a so-called stem structure, and the loop portion in the stem structure is a gene amplification product. It is designed to be complementary to a target region (for example, the base sequence region (2) described above). Furthermore, when the fluorescent agent and the non-fluorescent quencher are bonded to both ends of the nucleic acid, respectively, and the probe is released in the solution, a hairpin structure is formed, so that the fluorescent agent and the quencher interact with each other. The fluorescence is gone. However, when there is a gene amplification product in which a nucleic acid has a complementary base sequence, the loop part binds to the complementary base sequence, and as a result, the structure of the entire probe changes, and the fluorescent agent Since the quencher is separated from each other and the quenching effect of the quencher on the fluorescent agent is eliminated, the fluorescent agent emits the original fluorescence. By monitoring this increase in fluorescence intensity, the presence of the target base sequence can be detected. In the molecular beacon probe, the above-mentioned fluorescent agent and quencher label are usually labeled at the 5 ′ end of the nucleic acid with 6-carboxyfluorescein (6-FAM) or 6-carboxy-4,7,2 ′, 7 ′. Fluorescein-based fluorescent dyes such as -tetrachlorofluorescein (TET) and rhodamine-based fluorescent dyes such as 5-carboxytetramethylrhodsmine (TAMARA) are labeled, and at the 3 'end, 4- (4'-dimethylaminophenylazo) benzoic acid (DABCYL), etc. It can be performed by labeling a quencher (for example, Nature Biotechnology 1996 14: 303-308).

Tack - Man probe method can be used to be monitored by fluorescence formation of gene amplification products during the amplification process, a hybridization probe tack - Man probe (Taq-Man Probe) (Tack - Man (Taq-Man ) Is a registered trademark) and is a method for detecting genes (Experimental Medicine Vol.15 No.7 (extra number) p46-51,1997, etc.). The tack-man probe is a nucleic acid labeled with a fluorescein fluorescent dye (reporter dye) at the 5 ′ end and a rhodamine fluorescent dye (quencher dye) at the 3 ′ end. In a state where the reporter dye and the quencher dye are bound via the nucleic acid, the fluorescence of the reporter dye is suppressed by the quencher dye due to the Forster resonance energy. In contrast, when the primer and the tack-man probe anneal the nucleic acid complementary to the nucleic acid of the tack-man probe of the gene amplification product and the extension reaction proceeds, the 5 ′ → 3 ′ endonuclease activity of Taq DNA polymerase causes When the reporter dye at the 5 ′ end of the tack-man probe is detached from the quencher dye at the 3 ′ end, the fluorescence intensity of the suppressed reporter dye increases. The increase in fluorescence intensity by the reporter dye is proportional to the increase in gene amplification products having a base sequence complementary to the nucleic acid. By monitoring this increase in fluorescence intensity, the presence of the target base sequence can be detected in the gene amplification process (possible even after the gene amplification process is completed). The above-described fluorescent label in the Tac-Man probe is usually a fluorescein fluorescent dye such as 6-FAM or TET at the 5 ′ end of the nucleic acid, and a rhodamine fluorescent dye such as TAMARA at the 3 ′ end. Can be performed according to a conventional method (for example, Nucleic Acids Research 1993 21 (16): 3761-3766).

  The cycling probe method is a detection method using a combination of a chimeric probe composed of RNA and DNA and RNase H, and is a method capable of efficiently detecting a specific base sequence during or after gene amplification. The probe is labeled with a fluorescent dye (reporter) on the 5 ′ end side and with a quencher (quencher) on the 3 ′ side. In this state, the fluorescence is suppressed by the contact between the fluorescent dye and the quencher. However, after the RNA portion is cleaved by RNaseH after binding to a complementary amplification product, the probe emits strong fluorescence. By measuring the fluorescence intensity, the amount of the target gene amplification product can be monitored.

  In this detection method, when detecting a sapovirus, the target nucleic acid can be quantified and detected by the above-mentioned means. For example, it can be detected as qualitative information such as positive or negative without quantification. Is also possible. The detection information (quantitative value and qualitative information) of this gene amplification product etc. is used as an index to detect the desired sapovirus by associating it with the presence / absence of the sapovirus in the sample, and also with the abundance. Can do.

<Detection kit>
In the present invention, a sapovirus detection kit (hereinafter also referred to as the present detection kit) for carrying out the present detection method is also provided.

  In this detection kit, a gene amplification primer and / or a gene amplification product used for amplifying a conserved region of sapovirus by gene amplification means such as RT-PCR method or real-time PCR method is usually used. A probe for detecting the conserved region is included, but it is preferable that both the primer and the probe are included. A gene amplification primer is a nucleic acid that can be amplified as a gene amplification product by using a conserved region of a sapovirus gene or the like by using the gene amplification means. The nucleic acid probe for detection is a nucleic acid containing a base sequence complementary to a sequence corresponding to a base sequence such as a conserved region. As described above, a nucleic acid probe labeled with a fluorescent label, a dye label, an isotope label, or the like can be used as a nucleic acid probe for detection. However, particularly when a sapovirus is detected in a gene amplification process (real-time PCR method). When using the above, it is preferable to use the above TaqMan probe or a molecular beacon probe incorporated as a detection probe.

  The gene amplification primer and gene detection nucleic acid probe that can be included in the detection kit are as disclosed in the description of the detection method, and specific examples are also disclosed in the examples.

  Examples of the present invention will be disclosed below. However, the scope of the present invention is not limited by this embodiment.

<Preparation of primers and probes>
The detection of the sapovirus in the present Example was performed using the following gene amplification primers and gene detection nucleic acid probes whose base sequences were set using SEQ ID NOs: 1 to 16. These nucleic acids were chemically synthesized using an AB 13948 fully automatic nucleic acid synthesis and purification system (Applied Biosystems).

Primer for gene amplification [Forward-Primer]
F01: 5'-CCAGGCTCTCGCCACCTAC-3 '(SEQ ID NO: 17: equivalent to reference strains 5076 to 5094)
F02: 5'-CCAGGCTCTCGCTACCTAC-3 '(SEQ ID NO: 18: equivalent to 5079-5097 of AY603425 strain)
F03: 5'-TTTGGCCCTCGCCACCTAC-3 '(SEQ ID NO: 19: equivalent to 762-780 of AF294739 strain)
[Reverse-Primer]
R01: 5'-GCCCTCCATCTCAAACACTATTTTG-3 '(SEQ ID NO: 20: equivalent to reference strains 5154-5178)
R02: 5'-GCCCTCCATTTCAAACACTAATTTG-3 '(SEQ ID NO: 21: equivalent to 840-864 of AF294739 strain)

Probe for gene detection [Tack-Man probe]
5'-TGGTTYATAGGYGGTRCA-MGB-3 '(SEQ ID NO: 22: equivalent to reference strains 5102-5120)
(MGB: Minor Groove Binder (Tm enhancer))
As reagents for real-time PCR reaction, (1) the above-mentioned sense primers F01 to F03 prepared at 7 pmol / μl, (2) the above-mentioned antisense primers R01 to R02 prepared at 10 pmol / μl, and (3) 5 pmol / μl. 1 μl of each of the above-mentioned TaqMan probes prepared, (4) 12.5 μl of 2 × TaqMan Master mix (Applied Biosystems) and 1.5 μl of DW were mixed, and 5 μl of a plasmid solution or a cDNA solution described below was added.

  The Real-time PCR reaction was pretreated at 50 ° C. for 2 minutes and 95 ° C. for 10 minutes, and the reaction at 95 ° C. for 15 seconds and 60 ° C. for 1 minute was performed 45 times.

  The detection sensitivity of the present invention is the amplification region sequence of the Sapovirus G1a strain (SEQ ID NO: 23), the amplification region sequence of the G1b strain (SEQ ID NO: 24), the amplification region sequence of the G2a strain (SEQ ID NO: 25), and the amplification region sequence of the G2b strain. (SEQ ID NO: 26) and a G4 strain amplification region sequence (SEQ ID NO: 27) were examined using a dilution series of a plasmid vector. The lower limit of detection was 10 copies for all genotypes.

  Sapovirus, which is a cause of food poisoning or diarrhea, proliferates in human digestive organs and is excreted by being contained in feces, so frozen human stool was selected as a specimen. 1341 frozen stool was suspended in 4 volumes of TE buffer in a 2 ml tube to prepare a 20% emulsion preparation. This was centrifuged at 8000 rpm for 10 minutes at room temperature. 140 μl of the supernatant was collected and made into 50 μl of RNA purification solution using QIAamp virus RNA kit (QIAGEN). According to the usage method of SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen), 5 μl of the RNA purified solution was reacted at 42 ° C. for 2 hours to obtain a 20 μl cDNA solution. Of the 1341 frozen stool specimens, 4 (0.3%) were positive. Of the 4 cases that became positive, 3 cases (the target DNA concentration was judged to be too low) with a Ct value of 40 or more were compared with conventional RT-PCR (1) (Archives of Virology (2002) 147: 1445 -1451), only one case was positive. For the remaining 2 cases, sense primer (SV01F: 5'-TACRAHKCNTGGTTYATAGGTGGT-3 '(SEQ ID NO: 28) and antisense primer (SVG2aR: 5'-GCACTCGTTGGYCCAATGGT-3', SVG2b / dR: 5'-CGCTGTAGGRCCAATGGT-3 ' , SVG2cR: 5'-GCCGCGCTTGGTCCAATTGT-3 ') (SEQ ID NO: 29-31) was used to perform RT-PCR (2) to specifically detect G2 type, and confirmed that it was positive by agarose gel electrophoresis Each obtained band was excised and purified, and sequenced with the primers used for PCR.The PCR product of the sample that was positive in (1) had 98% homology with accession No. X86559, The PCR products of the samples that were positive in (2) were 97% homologous to AY237420 in both cases.

  As a result, it was confirmed that sapovirus can be detected by performing real-time PCR using probes and primers set based on a base sequence conserved in a specific region of the sapovirus genome.

It is drawing which showed the positional relationship of the base sequence of the gene of a sapovirus strain, and the primer probe used in the Example of this invention.

Claims (7)

The base sequence represented by SEQ ID NO: 5 as a cDNA of a sapovirus prototype (reference strain),
(1) In a base sequence corresponding to Nos. 5071 to 5094, a set of three or more + -strand nucleic acids (a-1) in which 15 to 24 bases are continuous is used as one gene amplification primer, and 5154 to 5178 Using a pair (b-2) of two or more negative-strand nucleic acids in which 15 to 25 bases are continuous in the base sequence corresponding to the number as the other gene amplification primer, or
(2) A group of three or more negative-strand nucleic acids (a-2) in which 15 to 24 bases are continuous in the base sequence corresponding to the numbers 5071 to 5094 is used as one gene amplification primer, and the numbers 5154 to 5178 Using a pair (b-1) of two or more + strand nucleic acids in which 15 to 25 bases are continuous in the base sequence corresponding to the above, as the other gene amplification primer,
A method of applying a gene amplification means to a gene obtained from a sample, and detecting a sapovirus in the sample using a gene detection probe obtained from the gene amplification product ,
(3) The above-mentioned three or more types of + strand nucleic acid sets (a-1) or − strand nucleic acid sets (a-2) are the same as the nucleic acids of SEQ ID NOS: 17, 18 and 19, respectively. Always included as a chain,
(4) The above-described two or more types of nucleic acid pairs (b-1) or -strand nucleic acid pairs (b-2) are the same as the nucleic acid pairs of SEQ ID NOS: 20 and 21, respectively. Always include as
(5) The nucleic acid probe for gene detection is a fluorescently labeled nucleic acid probe for gene detection including a + strand or − strand base sequence continuous with at least 10 bases among base sequences corresponding to 5101 to 5121 ,
A virus detection method characterized by the above. The virus detection method according to claim 1 , wherein the gene amplification primer is a gene amplification primer comprising a combination of the following forward primer (1) and reverse primer (2).
(1) As a forward primer, a set of nucleic acids having the nucleotide sequences of SEQ ID NOs: 17, 18 and 19 ;
(2) A set of nucleic acids having the nucleotide sequences of SEQ ID NOs: 20 and 21 as reverse primers . Fluorescently labeled for gene detection nucleic acid probe, Molecular Beacon probes (Molecular beacon Probe), or a hybridization probe that can be used to monitor by fluorescence formation during the amplification process of gene amplification products, claim The method for detecting a virus according to 1 or 2 . The virus detection method according to any one of claims 1 to 3 , wherein the virus detection method is a quantitative detection method of sapovirus. Analyte in the detection method is a stool sample, the detection method of the virus according to any one of claims 1-4. A virus detection kit for carrying out the virus detection method according to any one of claims 1 to 5 , comprising the following gene amplification primer and / or gene detection nucleic acid probe.
(1) The gene amplification primer is a gene amplification primer comprising a combination of the following forward primer (a) and reverse primer (b).
(A) As a forward primer, a set of nucleic acids having the nucleotide sequences of SEQ ID NO: 17, 18 and 19 ;
(B) As a reverse primer, a set of nucleic acids having the nucleotide sequences of SEQ ID NOs: 20 and 21 ;
(2) The nucleic acid probe for gene detection was continuous for at least 10 bases or more among the base sequences corresponding to the 5101 to 5121 base sequences represented by SEQ ID NO: 5 as the cDNA of the sapovirus prototype (reference strain) A fluorescently labeled nucleic acid probe for gene detection , which comprises a + strand or − strand base sequence . Fluorescently labeled for gene detection nucleic acid probe, Molecular Beacon probes (Molecular beacon Probe), or a hybridization probe that can be used to monitor a fluorescence formation of gene amplification products during the amplification process, claim 6. The virus detection kit according to 6 .