JP3847322B2 - Method for genotyping small spherical viruses - Google Patents

Description

  The present invention relates to a detection method and genotype identification method for small spherical viruses.

  Small round structured virus (SRSV) has been detected in patients with non-bacterial diarrhea and acute gastroenteritis (Non-patent document 1; Non-patent document 2). This virus is a positive single-stranded RNA virus of about 7,500 bp, and its electron microscope image has a diameter of 35 to 40 nm (Non-patent Document 3; Non-patent Document 4). The prototype was Norwalk virus, which was first isolated in 1968 in Ohio, USA, and its form was revealed in 1972 under an immunoelectron microscope (Non-patent Document 5). Thus, SRSVs that are morphologically similar to Norwalk virus but antigenically different are collectively referred to as Norwalk-like viruses. Regarding the gene structure of SRSV, ORF1 (5.4 kb), ORF2 (1.6 kb) and ORF3 (0.6 kb) encoding nonstructural protein, structural protein and protein with unknown function have been identified from the 5 ′ end (non-patented). Reference 3; Non-patent reference 4). SRSV is Geno Group I (Norwalk-like), Geno Group II (Snow mountain-like), Classical human calicivirus (Sapporo-like) It is classified into three types of groups.

  There have been 908 cases of non-bacterial gastroenteritis outbreaks in Japan over the five years since 1990, and the causative virus was identified in 360 cases, of which 330 were reported to be positive for SRSV. In addition, from January to November 1997, 198 cases of non-bacterial gastroenteritis outbreaks were reported, 152 cases except for 43 cases were food-borne, of which 71 cases were detected with SRSV (Non-patent document 6).

  Currently, detection of SRSV is mainly performed by an enzyme antibody method or a detection method using an immunomicroscope, using an affected patient's stool as a sample. The enzyme antibody method requires an antibody corresponding to various serotypes of SRSV and is expected to have a low practicality as a test because an increase in serotype is expected in the future. In addition, detection by an electron microscope is limited in facilities where it can be performed and it may be difficult to cope with a large amount of processing.

  In addition, since SRSV has a risk of causing non-bacterial diarrhea and gastroenteritis in humans in a wide range through food, it can rapidly remove trace amounts of virus from foods that can cause it before infection. There is also a need to establish a method that can detect at a high rate. However, when the SRSV infection source is food, the amount of virus is considered to be very small compared to stool, so there is a possibility that SRSV may not be detected by a method similar to the detection method using stool as a sample.

  Recently, detection based on a PCR (polymerase chain reaction) method in which a target gene fragment is amplified and detected as a method that can be measured even with a small amount of virus in a sample has been reported for SRSV. The amplification primer is mainly set to amplify a part of RdRp (RNA-dependent RNA polymerase) region present in ORF1 (open reading frame 1) (Non-patent document 7; Non-patent document 8; Non-patent document) 9; Non-patent document 10; Non-patent document 11).

At present, there is no separation and growth method using cultured cells or experimental animals for SRSV, and methods for identifying viruses from humans include enzyme antibody method using patient stool as a sample, electron microscopy or Western blotting. The method used is mainly performed (Non-patent document 12; Non-patent document 13; Non-patent document 14; Non-patent document 15; Non-patent document 16). However, the method using an antibody requires an antibody corresponding to various serotypes of SRSV, and the method using an electron microscope is limited in facilities where it can be performed, and it is difficult to deal with a large amount of processing. is there.
J. Clin. Microbiol. 32: 642-648 (1994) J. Clin. Microbiol. 27: 1728-1733 (1989) Science 259: 516-519 (1993) Science 250: 158-1583 (1990) J. Virol. 10: 1075-1081 (1972) Infectous Agents Surveillance Report. 19: 1-6 (1998) J. Clin. Microbiol. 30: 2529-2534 (1992) Arch. Virol. 135: 185-192 (1994) J. Clin. Microbiol. 33: 64-71 (1995) J. Clin. Microbiol. 35: 570-577 (1997) Microbiol. Immunol. 42: 439-446 (1998) J. Med. Virol. 2: 97-108 (1987) J. Med. Virol. 17: 127-133 (1985) J. Clin. Microbiol. 22: 274-278 (1985) J. Clin. Microbiol. 24: 456-459 (1986) J. Clin. Microbiol. 10: 730-736 (1979)

  As a result of comparison of the already reported SRSV base sequences, the present inventors have many base sequence variations in ORF1, and the PCR detection method using conventional primers based on the base sequence of ORF1 gives false negatives. I found that there is a point to improve such as high possibility.

  Accordingly, an object of the present invention is to provide a method for detecting SRSV that does not have the above-described problems.

  Another object of the present invention is to provide a rapid and simple method for identifying the SRSV genotype.

  The present inventors set based on the base sequence of the region conserved in each of genogroups I and II in the base sequence including the region including the 3 ′ end of ORF1 to the 5 ′ end region of ORF2. By performing PCR using a primer, particularly a reverse transcription PCR (RT-PCR) reaction primer for detecting each genogroup, SRSV can be specifically detected, and the 5 ′ end region of ORF2 It was found that the SRSV genotype can be identified by performing phylogenetic analysis based on the base sequence, and the present invention was completed.

  Therefore, the present invention synthesizes cDNA from RNA obtained from a sample by reverse transcriptase, performs PCR using this as a template, and detects an amplified product generated by PCR. The primer used in the PCR comprises about 30 bp of the 3 'end of the open reading frame 1 (ORF1) region of the SRSV genome and about 20 bp of the 5' end of the open reading frame 2 (ORF2) of the SRSV genome. A first primer set based on the base sequence conserved in the region to be included, and a base sequence conserved in the region of base numbers from about 292 to about 313 counting from the 5 ′ end of ORF2. The second primer, the first primer and the second primer are It provides a method for detecting SRSV characterized (detection method of the present invention) to which is set to be capable of amplifying the nucleotide sequence between.

  In the detection method of the present invention, preferably, cDNA synthesis is performed using a primer having the base sequence shown in SEQ ID NO: 1, and PCR is performed in two stages, and the first stage PCR is shown in SEQ ID NO: 1. A primer having a base sequence and a primer having a base sequence shown in SEQ ID NO: 2 are used, and a primer having a base sequence shown in SEQ ID NO: 1 and a primer having a base sequence shown in SEQ ID NO: 3 are used in the second stage PCR. Or the synthesis of the cDNA is carried out using a primer having the base sequence shown in SEQ ID NO: 4, and the PCR is carried out in two stages, and the base sequence shown in SEQ ID NO: 4 in the first stage PCR And a primer having the base sequence shown in SEQ ID NO: 5 are used, and the second-stage PCR has the base sequence shown in SEQ ID NO: 4 A primer having a nucleotide sequence shown in primer SEQ ID NO: 6 is used.

  The present invention also provides a molecule based on a base sequence that can be identified from the range of base numbers from about 1 to about 300, counting from the 5 ′ end of the ORF2 region of the SRSV genome, by molecular phylogenetic analysis. A method for identifying the genotype of SRSV (the identification method of the present invention) characterized by identifying the genotype of SRSV by performing phylogenetic analysis is provided.

  In the identification method of the present invention, preferably, the base sequence to be subjected to molecular phylogenetic analysis is set so that cDNA can be synthesized from RNA obtained from a sample by reverse transcriptase and the above base sequence can be amplified using this cDNA as a template. PCR was performed using the prepared primers, and the base sequence of the amplification product generated by PCR was determined. Further, preferably, the synthesis of the cDNA is performed using a primer having the base sequence shown in SEQ ID NO: 1, and PCR is performed in two stages, and the base sequence shown in SEQ ID NO: 1 is added to the first stage PCR. Whether the primer having the base sequence shown in SEQ ID NO: 3 and the primer having the base sequence shown in SEQ ID NO: 3 are used in the second-stage PCR. Alternatively, the cDNA synthesis is performed using a primer having the base sequence shown in SEQ ID NO: 4, and PCR is performed in two stages, and the primer having the base sequence shown in SEQ ID NO: 4 in the first stage PCR And a primer having the base sequence shown in SEQ ID NO: 5, and a primer having the base sequence shown in SEQ ID NO: 4 in the second stage PCR, A primer having a nucleotide sequence shown in column number 6 is used.

  The present invention provides a method for detecting SRSV rapidly and at a high rate, capable of detecting trace amounts of viruses such as viruses in foods. In addition, a rapid and simple method for identifying the SRSV genotype is provided.

<1> Detection method of the present invention The detection method of the present invention is a method that makes it possible to detect SRSV with high sensitivity and specificity, that is, cDNA is synthesized from RNA obtained from a sample by reverse transcriptase, and this is used as a template. And detecting the amplification product generated by PCR, wherein the primers used in the PCR are about 30 bp of the 3 ′ end of the ORF1 region of the SRSV genome and the SRSV genome. A first primer set based on a base sequence conserved in a region containing about 20 bp of the 5 ′ end of ORF2 of ORF2, and a region of base numbers about 292 to about 313 counted from the 5 ′ end of ORF2 A second primer set based on the base sequence of the first primer and the second primer between them It is set so that it can amplify the base sequence of SRSV.

The sample is not particularly limited as long as it contains or may contain SRSV, but examples include clinical materials, virus cultures separated from these clinical materials, and foods such as oysters. Can do. RNA can be prepared from these samples by a known method such as extraction. A method for synthesizing cDNA from RNA using reverse transcriptase is also known. PCR can be performed according to a known PCR method using this cDNA as a template. For cDNA synthesis, it is preferable to use the first primer and the second primer that are complementary to RNA, because cDNA synthesis and subsequent PCR amplification can be performed in the same reaction system.

  The first primer and the second primer are set so that the base sequence between them can be amplified, that is, a sense primer and an antisense primer.

  The first primer is preferably set based on the base sequence conserved in the region containing about 30 bp of the 3 'end of the ORF1 region of the SRSV genome and about 20 bp of the 5' end of the ORF2 of the SRSV genome. The second primer is preferably set based on the base sequence conserved in the region of base numbers from about 292 to about 313 counted from the 5 'end of ORF2.

  Selection of a conserved base sequence in the specific region and setting of primers may be performed by methods known to those skilled in the art with reference to the known base sequence of the SRSV genome. The selection of the conserved base sequence and the setting of the primer are made more efficient by being performed based on computer search.

  Primers may be set specifically for each SRSV genogroup. In this way, the number of base mutations in the primer can be reduced and the specificity can be increased. In addition, the genogroup can be determined quickly only by detecting the PCR amplification product without decoding the base sequence of the PCR amplification product. Can do.

  The primer may be set as a mixed primer obtained by mixing a plurality of primers with respect to one or both of the sense primer and the antisense primer. When a base mutation exists in the primer setting part, the detection efficiency can be increased by using a mixed primer.

  Preferable examples of the primer include, in the case of genogroup I, a primer having the base sequence shown in SEQ ID NO: 1 or 3 and a primer having the base sequence shown in SEQ ID NO: 2, and in the case of genogroup II And a primer having the base sequence shown in SEQ ID NO: 4 or 6 and a primer having the base sequence shown in SEQ ID NO: 5.

  Amplification by PCR may be performed in two stages. In the case of genogroup I, for example, the first round is performed using a primer having the base sequence shown in SEQ ID NO: 1 and a primer having the base sequence shown in SEQ ID NO: 2. After performing PCR, the second PCR may be performed using a primer having the base sequence shown in SEQ ID NO: 1 and a primer having the base sequence shown in SEQ ID NO: 3. In the case of Geno Group II, for example, after performing the first PCR using a primer having the base sequence shown in SEQ ID NO: 4 and a primer having the base sequence shown in SEQ ID NO: 5, The second PCR may be performed using a primer having the nucleotide sequence shown and a primer having the nucleotide sequence shown in SEQ ID NO: 6. By performing amplification in two stages, it becomes possible to amplify the target base sequence more specifically.

PCR can be performed according to a known method by synthesizing cDNA from RNA obtained from a sample using reverse transcriptase, using this cDNA as a template, and using the above primers, and using reverse transcription PCR (RT-PCR) in the same reaction system. Acquisition of cDNA and amplification of DNA may be performed. In this case, it is preferable to use a primer complementary to RNA among the primers used in PCR for the reaction by reverse transcriptase. For example, if the above two-step PCR is employed, the primer shown in SEQ ID NO: 1 and The primer shown in SEQ ID NO: 4 is used.

  The amplification product can be detected by a known analysis method such as agarose gel electrophoresis and ethidium bromide staining. Usually, an amplification product having a length predictable from the known base sequence of the SRSV genome and the base sequence of the primer used is detected.

<2> Identification method of the present invention The identification method of the present invention is selected from the range of base numbers of about 1 to about 300 from the 5 'end of the ORF2 region of the SRSV genome in order to identify the genotype of SRSV. Molecular phylogenetic analysis is performed based on a base sequence (hereinafter also referred to as a base sequence of the ORF2N terminal region) that can identify the genotype of SRSV by molecular phylogenetic analysis.

  The method for obtaining the base sequence of the ORF2N terminal region is not particularly limited, but when the base sequence of the ORF2N terminal region is obtained from a sample containing an unidentified SRSV genotype, the following method is preferred. That is, cDNA is synthesized from RNA obtained from a sample by reverse transcriptase, and PCR is performed using primers set so as to amplify a base sequence including the base sequence of the ORF2N terminal region of the SRSV genome using this cDNA as a template. This is a method for determining the base sequence of an amplification product generated by PCR. Preparation of RNA from a sample, preparation of cDNA, and PCR can be performed as described above for the detection method of the present invention.

  As a preferable example of a primer set so as to be able to amplify a base sequence including the base sequence of the ORF2N terminal region, for Genogroup I, a primer having the base sequence shown in SEQ ID NO: 1 or 3 and a base shown in SEQ ID NO: 2 The primer which has a sequence is mentioned, About Genogroup II, the primer which has the base sequence shown to sequence number 4 or 6 and the primer which has the base sequence shown to sequence number 5 are mentioned.

  Amplification by PCR may be performed in two stages. In the case of genogroup I, for example, the first round is performed using a primer having the base sequence shown in SEQ ID NO: 1 and a primer having the base sequence shown in SEQ ID NO: 2. After performing PCR, the second PCR may be performed using a primer having the base sequence shown in SEQ ID NO: 1 and a primer having the base sequence shown in SEQ ID NO: 3. In the case of Geno Group II, for example, after performing the first PCR using a primer having the base sequence shown in SEQ ID NO: 4 and a primer having the base sequence shown in SEQ ID NO: 5, The second PCR may be performed using a primer having the nucleotide sequence shown and a primer having the nucleotide sequence shown in SEQ ID NO: 6. By performing amplification in two stages, it becomes possible to amplify the target base sequence more specifically.

  When cDNA is obtained and DNA is amplified in the same reaction system by reverse transcription PCR (RT-PCR), it is preferable to use a primer complementary to RNA for the reaction by reverse transcriptase. For example, if the above two-step PCR is employed, the primer shown in SEQ ID NO: 1 and the primer shown in SEQ ID NO: 4 are used.

  The amplification product can be obtained by a known method such as separation by agarose gel electrophoresis, extraction from the gel, and extraction purification. Usually, an amplification product having a length predictable from the known base sequence of the SRSV genome and the base sequence of the primer used is obtained.

The base sequence of the amplification product can be determined by a known method. Among the determined base sequences, the base sequence of the ORF2N terminal region is used for molecular system analysis. The base sequence of the ORF2N terminal region is a part where a significant result is obtained in the molecular phylogenetic analysis, and can vary within the above range depending on the range of the genotype targeted for the molecular phylogenetic analysis, It is easy for those skilled in the art to select an appropriate part. For example, in the case of molecular phylogenetic analysis of Geno Group I, among the nucleotide sequences registered in GenBank with Accession No. M87761, a 230 bp nucleotide sequence of nucleotide numbers 5400-5629 and a 187 bp nucleotide sequence of nucleotide numbers 5441-5627 In the case of Geno Group II, among the base sequences registered in GenBank with Accession No. X86557, there are 216 bp base sequences of base numbers 5127 to 5342 and 187 bp base sequences of base numbers 5156 to 5342. It is done.

  In the identification method of the present invention, molecular phylogenetic analysis based on the base sequence of the ORF2N terminal region can be performed by a known molecular phylogenetic analysis method. Examples of such methods include the Higgins method, the UPGMA method using the two-parameter method, and the NJ method. Software for performing molecular phylogenetic analysis using these methods is commercially available (DNASIS (Hitachi Software Engineering), SINCA (Fujitsu), etc.).

  The genotype can be determined based on whether or not the genotype shows a homology of a certain value or more with a standard strain of each genotype in the molecular phylogenetic analysis or is distinguished from other genotype clusters with a probability of a certain value or more. The constant value may be a value that allows each strain belonging to each genotype to form a single cluster in the phylogenetic tree obtained by molecular phylogenetic analysis. For example, the value is such that a single gene forms a cluster without crossing with a probability of 100% in the bootstrap method. More specifically, a value of 80% can be mentioned.

  EXAMPLES Next, the present invention will be described in detail with reference to examples. However, the following examples are given only as an aid for obtaining specific recognition about the present invention, and thereby the scope of the present invention is limited in any way. It is not a thing.

[Example 1] Detection of SRSV (1) Setting of primer The primer is a nucleotide sequence (DS395: U04469, NV: M87661, registered in GenBank or EMBL as the nucleotide sequence of the genome of SRSV already detected from humans). KY-89 / 89 / J: L23829, SOV: L07418, BR: X76716, LD: X86557, CW: U46500, TV: U02030, OTH-25 / 89 / J: L23830, MX: U22498, AL: U46039, SMA: L23831, HW: U07611, MS: X81879, MI1 / 94 / Japan:
AB005259).

  That is, with respect to the primers of Geno Group I, based on the base sequences of DS395, NV, KY-89 / 89 / J and SOV, antisense primer (G1 / R1) 5′CCAACCCARCCATTRTACATTT3 ′ (SEQ ID NO: 1), 1st Sense primer (G1 / F1) 5′CTGCCCGAATTYGTAAATGAT3 ′ (SEQ ID NO: 2) for the first PCR (1st PCR) reaction, and sense primer (G1 inside G1 / F1 for the second PCR (2nd PCR) reaction) / F2) 5′AATGATGATGGCGTCTAAGGA3 ′ (SEQ ID NO: 3) was synthesized. Genogroup II primers are antisense based on the base sequences of BR, LD, CW, TV, OTH-25 / 89 / J, MX, AL, SMA, HW, MS, MI1 / 94 / Japan. Primer (G2 / R1) 5'TGCATAACCATTRTACATTCT3 '(SEQ ID NO: 4), sense primer (G2 / F1) for 1st PCR reaction, 5'GTGGGAGGGCGATCGCAATCT3' (SEQ ID NO: 5), and inside of G2 / F1 for 2nd PCR reaction An oligonucleotide having the base sequence of the sense primer (G2 / F3) 5′TTGTGAATGAAGATGGCGTCGA3 ′ (SEQ ID NO: 6) was synthesized. The relationship between the gene structure of SRSV and the position of the primer is as shown in FIG.

The base sequences of G1 / F1, G1 / R1 and G1 / F2 are the base sequences registered in GenBank with Accession No. M87661, base numbers 5342 to 5362, 5671 to 5650 and 5357 to 5377, respectively.
Corresponds to the nucleotide sequence of. In addition, the base sequences of G2 / F1, G2 / R1 and G2 / F3 are the base numbers 5047 to 5067, 5384 to 5364, and 5079 to 5100 in the base sequence registered in GenBank with Accession No. X86557, respectively. Corresponds to an array.

  By using these primers, for Genogroup I (DS395, NV, KY-89 / 89 / J and SOV), the base sequence corresponding to base numbers 5242 to 5671 is amplified in the base sequence of M87661. Primer was set. For Geno Group II (BR, LD, CW, TV, OTH-25 / 89 / J, MX, AL, SMA, HW, MS, MI1 / 94 / Japan), base numbers 5047 to 5384 in the base sequence of X86557 A base sequence corresponding to is amplified.

  In addition, G1 / F2 and G2 / F3 were DIG-labeled for use as probes for Southern hybridization.

(2) Detection of SRSV from food (oyster) Oyster was selected as a sample because it is considered one of the sources of SRSV infection in Japan. Five oyster midgut glands were excised and pooled into one specimen. Add an equal amount of TE (10 mM Tris-HCl, 1 mM EDTA (pH 8.0), Wako Pure Chemicals), homogenize with a stomacher to prepare a 50% emulsion, and add this to a 50 ml tube. Transfer and centrifuge at 3500 rpm for 35 minutes at 4 ° C. Dispense 2 ml of the centrifugal supernatant into a 50 ml tube, add 8 ml of TE to make a 10% emulsion preparation, add 10 ml of HCFC-141b (Daikin Industries) and mix at 4 ° C, 3500 rpm. Centrifuge for 35 minutes. Dispense 500 μl of 30% sucrose into Opti Seal (BECKMAN), overlay 4 ml of HCFC-141b-treated preparation, perform ultracentrifugation at 4 ° C. and 40000 rpm for 2 hours, and add 200 μl to the precipitate. TE was dispensed to dissolve the precipitate. 500 μl of lysate was added to the RNA extract containing Sumitest-R Kit (Sumitomo Metal Industries) and 50 μl of CHCl ₃ in a 1.5 ml tube with a screw cap, and RNA was extracted according to the kit usage. . Oyster homogenate is purified by ultracentrifugation using HCFC-141b treatment and 30% sucrose density gradient, and then RNA synthesis kit reagents can be used to remove cDNA synthesis and PCR inhibitors .

Extracted and dried RNA is dissolved in 60 μl of RNase inhibitor (40 U / μl) mixed TE and dissolved, and then 10 μl of the lysate is dispensed into a separate tube to prepare G1 / R1 or 100 pmol / μl. 1 μl of G2 / R1 was added to a tube into which 10 μl of the lysate was dispensed, mixed, heated at 100 ° C. for 2 minutes, and rapidly cooled in ice. To this, 2 μl of 10 × TaqGold Buffer, 2 μl of 10 mM dNTPs, 1 μl of 200 U / μl M-MLV-derived reverse transcriptase, 1 μl of 40 U / μl RNase inhibitor and 3 μl of sterilized distilled water were added at 37 ° C. for 60 minutes. CDNA was synthesized under the following reaction conditions. For genogroup I, the synthesized cDNA was 10 × TaqGold Buffer 4.5 μl, 10 mM dNTPs 0.5 μl, 100 pmol G1 / F1 0.5 μl, 100 mM G1 / R1
1st PCR was performed by adding 5 μl to a PCR reaction solution in which 0.25 μl, 5 U / μl TaqGold 0.25 μl, and sterilized distilled water 39 μl were mixed. The PCR reaction was performed at 95 ° C for 15 minutes, followed by 40 reactions of 94 ° C for 1 minute, 55 ° C for 2 minutes, 72 ° C for 3 minutes, and then heated at 72 ° C for 7 minutes. For Geno Group II, G1 / F1 was replaced with G2 / F1, and G1 / R1 was replaced with G2 / R1.

For Genogroup I 2nd PCR, 10 × TaqGold Buffer 4.5 μl, 10 mM dNTPs 1 μl, 100
5 μl of the 1st PCR product was added to the reaction mixture in which 0.5 μl of pmol / μl G1 / F2, 0.5 μl of 100 pmol / μl G1 / R1, 0.25 μl of 5 U / μl TaqGold, and 39 μl of sterile distilled water were mixed. For Geno Group II, G1 / F2 was replaced with G2 / F3, and G1 / R1 was replaced with G2 / R1. The conditions for 2nd PCR were the reaction conditions for 1st PCR.

  When SRSV is present in the sample, a PCR product of 333 bp is obtained for the 1st PCR of Geno Group I, a 318 bp PCR product for the 2nd PCR, a PCR product of 338 bp is obtained for the 1st PCR of Geno Group II, and a 300 bp PCR product is obtained for the 2nd PCR. It is done.

  The PCR reaction solution was electrophoresed on a 3% agarose gel for each genogroup, nucleic acid stained with ethidium bromide, and the PCR amplification product was detected by ultraviolet rays.

  Of the 85 oysters collected from the pool of the 5 oyster midgut glands used in this example, 35.3% of the oysters were positive. The breakdown of positive samples is 5 samples (5.9%) positive only for genogroup I, 14 samples (16.5%) positive only for genogroup II, 11 samples positive for both genogroups I and II (12.9%).

  Southern hybridization was carried out for confirmation of the positive 2nd PCR product. That is, the PCR product was transferred to the membrane from the gel on which the 1st PCR product was run. Prehybridization was performed at 55 ° C, and hybridization was performed at 37 ° C. In the PCR reaction, G1 / F2 labeled with DIG was used for genogroup I, and G2 / F3 labeled with DIG was used for genogroup II. After hybridization, the plate was washed twice with a washing solution at 37 ° C. (0.1 × SSC, 1% SDS), and then reacted according to the instructions of the DIG labeling kit. A sample in which hybridization with the labeled probe was confirmed was positive for SRSV (G1, G2 or G1G2 mixed). As a result, all positive specimens were confirmed by Southern hybridization.

  In confirmation of the band by electrophoresis of the PCR product, almost no non-specific band was observed, and only the target band was detected. In the electrophoresis after the 1st PCR reaction, all samples whose target band could be confirmed by the 2nd PCR reaction were positive for the hybridization of the 1st PCR product, even though the target band was not visually confirmed.

  Therefore, it was confirmed that SRSV can be detected by amplifying the base sequence by PCR using primers set based on the base sequence conserved in a specific region of the SRSV genome and examining the presence or absence of the amplified product.

[Example 2] Identification of genogroups and genotypes by molecular phylogenetic analysis In Example 1, the 2nd PCR product that was positive by Southern hybridization was analyzed using the QIAquick PCR purification kit (QIAGEN). Unreacted primers and dNTPs were removed, and the DNA concentration was adjusted with sterilized distilled water so as to be 5 ng / μl. For genogroup I, G1 / F2 was used on the sense side and G1 / R1 PCR primers were used on the antisense side, and G2 / F3 on the sense side and G2 on the antisense side. The nucleotide sequence was determined with a 373A DNA autosequencer (PERKIN ELMER) using a dideoxyterminator cycle sequence kit (PERKIN ELMER) using the / R1 PCR primer.

  Regarding the SRSV nucleotide sequence, one sample and one genotype, Genogroup I is a 230 bp nucleotide sequence excluding 23 bp from the 3 ′ end of G1 / F2 and 20 bp from the 3 ′ end of G1 / R1. Genogroup II determines the base sequence of 216 bp excluding 27 bp from the 3 'end of G2 / F3 and 23 bp from the 3' end of G2 / R1. Used for molecular phylogenetic analysis. These genogroups correspond to base numbers 5400 to 5629 among the genogroup I registered in GenBank with Accession No. M87661, and genogroup II is registered with GenBank with Accession No. X86557. It corresponds to the base numbers 5127 to 5342 in the base sequences. With respect to the analyzed base sequence, molecular phylogenetic analysis is performed together with the base sequence of the region corresponding to the base sequence registered in GenBank or EMBL of SRSV already detected in humans, and genogroup and genotype are identified. It was.

The phylogenetic tree is created using UPGMA and NJ (SINCA, FUJITSU) software using the 2-parameter method, and the systematic evaluation of the created phylogenetic tree using the Bootstrap method (SINCA) Went.

  As a result of the determination of the base sequence, one of 16 samples in Geno Group I and 6 of 25 samples in Geno Group II could not be completely determined. In these analyzed base sequence charts, similar peaks of two or more bases overlapped at the same site.

  A phylogenetic tree was created using the base sequence obtained from the positive specimen obtained in Example 1 and the base sequence of the human-derived SRSV (FIG. 2). In FIG. 2, those derived from the specimen are underlined. As a result, Genogroups I and II were classified with 100% probability. When the probability of classification by the bootstrap method is 80%, the 16 samples that were positive in Geno Group I in Example 1 were 1 Norwalk type sample, 4 Southampton type samples, and 10 Ciba type samples. And Hjgl1 specimens whose types were undecided. Similarly, 19 specimens used for analysis among 25 specimens that were positive in Geno Group II were classified into 15 Mexican-type specimens, 2 47-type specimens, and 2 Hawaii-type specimens.

  Therefore, it was confirmed that the genotype of SRSV can be identified by performing molecular phylogenetic analysis based on the base sequence of the specific region at the 5 'end side in the ORF2 region of the SRSV genome. It was also confirmed that genogroup classification was possible by the SRSV detection method in which PCR was performed using primers specific to genogroup.

The relationship between the gene structure of SRSV and the position of the primer is shown. The result of molecular phylogenetic analysis is shown.

Claims (4)

Molecular phylogenetic analysis based on the base sequence from which the genotype of SRSV can be identified by molecular phylogenetic analysis selected from the range of base numbers 1 to 300 counted from the 5 ′ end of the ORF2 region of the small spherical virus (SRSV) genome A method for identifying the genotype of SRSV, characterized by identifying the genotype of SRSV. Amplification generated by PCR by synthesizing cDNA from the RNA obtained from the sample using reverse transcriptase, performing PCR using the cDNA as a template, using primers set to amplify the base sequence The identification method according to claim 1, wherein the identification method is obtained by determining a base sequence of a product. Synthesis of the cDNA is performed using a primer having the base sequence shown in SEQ ID NO: 1, and the PCR is performed in two stages, and the primer and sequence having the base sequence shown in SEQ ID NO: 1 in the first stage PCR A primer having the base sequence shown in No. 2 is used, and a primer having the base sequence shown in SEQ ID No. 1 and a primer having the base sequence shown in SEQ ID No. 3 are used in the second stage PCR. 2. The identification method according to 2. Synthesis of the cDNA is performed using a primer having the base sequence shown in SEQ ID NO: 4, and the PCR is carried out in two stages, and the primer and sequence having the base sequence shown in SEQ ID NO: 4 in the first stage PCR The primer having the base sequence shown in No. 5 is used, and the primer having the base sequence shown in SEQ ID No. 4 and the primer having the base sequence shown in SEQ ID No. 6 are used in the second stage PCR. 2. The identification method according to 2.

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