JP2008079576A - Oligonucleotide set and use thereof - Google Patents

Abstract

An oligonucleotide set suitable for rapidly and comprehensively detecting two or more types of diarrhea-causing viruses.
SOLUTION: Adenovirus hexon gene, enterovirus 5 'untranslated region, herpes simplex virus type I DNA polymerase gene, herpes simplex virus type II glycoprotein gene, VP4 gene of group A rotavirus, group C rotavirus Polynucleotides encoding two or more kinds of regions selected from the VP4 gene, astrovirus protease gene, hepatitis A virus 5 'untranslated region, coronavirus surface glycoprotein gene and Aichi virus VP1 gene, Two or more diarrhea-causing viruses are rapidly and comprehensively detected by using an oligonucleotide set including an oligonucleotide that hybridizes with a part thereof.
[Selection figure] None

Description

  The present invention relates to an oligonucleotide set and use thereof, and more particularly to an oligonucleotide set and use thereof for detecting a diarrhea-causing virus.

Conventionally, as a method for detecting microorganisms such as bacteria and viruses, various methods such as a morphological examination method using an electron microscope, a cell culture method, a serological method, an antigen detection method, a PCR (Polymerase Chain Reaction) method, etc. The method is known. Among these, the PCR method is applicable to microorganisms that are difficult or impossible to cultivate, has the advantage that the time required for detection is shorter than other methods, and the detection sensitivity is high. . In addition, by using a multiplex PCR method in which PCR is performed using several types of primers, a plurality of types of microorganisms can be detected. For these reasons, the present inventors have focused on multiplex PCR as a detection method targeting a plurality of types of microorganisms that cause food poisoning including diarrhea symptoms. Has already developed a primer set that can be amplified by (eg, Patent Document 1). According to the primer set of Patent Document 1, a plurality of types of microorganisms that cause food poisoning including diarrhea symptoms can be comprehensively detected by a single gene detection operation.
JP 2003-164282 A

  However, although the above-mentioned Patent Document 1 discloses primers for bacteria and protozoa as microorganisms that cause food poisoning including diarrhea symptoms, a primer set capable of detecting diarrhea-causing viruses is disclosed. Is not specifically described. In addition, as a method for detecting a virus caused by diarrhea, at present, emphasis is placed on detecting a single virus, and its usefulness is limited. In addition, multiplex PCR is also used for simultaneous detection of various viruses. However, when more types of diarrhea-causing viruses are detected, the number of primers used increases, so the reaction conditions must be set. It becomes difficult. For this reason, it is difficult to design a primer set capable of comprehensively detecting a plurality of types of diarrhea-causing viruses with a single gene detection operation. Nucleotide sets such as primers and probes that are truly excellent for detection have not yet been found.

  Therefore, the present invention provides an oligonucleotide set, oligonucleotide, solid-phase carrier, detection kit, detection method, and diarrhea diagnosis method suitable for rapidly and comprehensively detecting two or more types of diarrhea-causing viruses. The purpose is to provide.

  The present inventors include adenovirus hexon gene, enterovirus 5 'untranslated region, herpes simplex virus type I DNA polymerase gene, herpes simplex virus type II glycoprotein gene, group A rotavirus VP4 gene, group C Focusing on rotavirus VP4 gene, astrovirus protease gene, hepatitis A virus 5 'untranslated gene, coronavirus surface glycoprotein gene and ichivirus VP1 gene, we searched specific sequences for each gene. However, the inventors have found that an oligonucleotide set containing two or more specific primer pairs capable of specifically amplifying these genes can be constructed significantly, and the present invention has been completed. That is, according to the present invention, the following means are provided.

According to one aspect of the invention, an oligonucleotide set for detecting a diarrhea-causing virus comprising:
Adenovirus hexon gene, enterovirus 5 'untranslated region, herpes simplex virus type I DNA polymerase gene, herpes simplex virus type II glycoprotein gene, VP4 gene of group A rotavirus, VP4 gene of group C rotavirus, A polynucleotide encoding at least two regions selected from an astrovirus protease gene, a 5 ′ untranslated region of hepatitis A virus, a surface glycoprotein gene of coronavirus, and a VP1 gene of Aichi virus, or a part thereof An oligonucleotide set is provided that includes oligonucleotides that hybridize.

  The oligonucleotide set of the present invention may contain 5 or more kinds of the oligonucleotides, or may contain 8 or more kinds of the oligonucleotides. Furthermore, the oligonucleotide set may detect two or more types of diarrhea-causing viruses at the same time.

In the oligonucleotide set of the present invention, the following (a) to (j):
(A) a base sequence set forth in SEQ ID NO: 1, a base sequence set forth in SEQ ID NO: 2, a base sequence set forth in SEQ ID NO: 23, a base sequence set forth in SEQ ID NO: 24, and those in an adenovirus hexon gene One or more oligonucleotides targeting a region sandwiched by any two base sequences of the complementary sequences of (b) The base sequence and sequence set forth in SEQ ID NO: 3 in the 5 ′ untranslated region of enterovirus One or more oligonucleotides targeting a region sandwiched by any two base sequences of the base sequence described in No. 4 and the base sequence described in SEQ ID NO: 25 and their complementary sequences (c ) The nucleotide sequence set forth in SEQ ID NO: 5 in the herpes simplex virus type I DNA polymerase gene, the nucleotide sequence set forth in SEQ ID NO: 6, One or more oligonucleotides that target a region sandwiched by any two base sequences of the base sequence set forth in column number: 26, the base sequence set forth in SEQ ID NO: 27, and their complementary sequences ( d) The base sequence set forth in SEQ ID NO: 7, the base sequence set forth in SEQ ID NO: 8, the base sequence set forth in SEQ ID NO: 28, the base set forth in SEQ ID NO: 29 in the herpes simplex virus type II glycoprotein gene One or more oligonucleotides targeting a region sandwiched between any two of the sequences and their complementary sequences (e) the base set forth in SEQ ID NO: 9 in the VP4 gene of group A rotavirus Sequence, base sequence described in SEQ ID NO: 10, base sequence described in SEQ ID NO: 11, base sequence described in SEQ ID NO: 12, SEQ ID NO: 30 1 type or 2 types or more of oligonucleotides which target the area | region pinched | interposed by any two base sequences among the base sequence as described in SEQ ID NO: 31, and their complementary sequences (f) Group C In the rotavirus VP4 gene, the base sequence described in SEQ ID NO: 13, the base sequence described in SEQ ID NO: 14, the base sequence described in SEQ ID NO: 32, the base sequence described in SEQ ID NO: 33, and their complementary sequences 1 or 2 or more types of oligonucleotides targeting a region sandwiched between any two of the nucleotide sequences (g) The nucleotide sequence set forth in SEQ ID NO: 15 in the protease gene of astrovirus, described in SEQ ID NO: 16 Nucleotide sequence of SEQ ID NO: 34, nucleotide sequence of SEQ ID NO: 35, nucleotide sequence of SEQ ID NO: 36 , One or more oligonucleotides targeting a region sandwiched by any two nucleotide sequences of the nucleotide sequence set forth in SEQ ID NO: 37 and their complementary sequences (h) 5 ′ of hepatitis A virus Of the base sequence set forth in SEQ ID NO: 17 in the untranslated region, the base sequence set forth in SEQ ID NO: 18, the base sequence set forth in SEQ ID NO: 38, the base sequence set forth in SEQ ID NO: 39, and their complementary sequences One or more oligonucleotides targeting a region sandwiched by any two nucleotide sequences (i) The nucleotide sequence described in SEQ ID NO: 19 in the coronavirus surface glycoprotein gene, described in SEQ ID NO: 20 Any of the base sequence of SEQ ID NO: 40, the base sequence of SEQ ID NO: 41, and their complementary sequences One or more oligonucleotides targeting a region sandwiched between two nucleotide sequences (j) the nucleotide sequence of SEQ ID NO: 21 in the VP1 gene of Aichivirus, the nucleotide sequence of SEQ ID NO: 22 One or more oligonucleotides targeting a region sandwiched by any two base sequences of the base sequence set forth in SEQ ID NO: 42, the base sequence set forth in SEQ ID NO: 43, and their complementary sequences Two or more kinds of oligonucleotides selected from the group consisting of:

In the oligonucleotide set of the present invention,
The one or more oligonucleotides of (a) are the nucleotide sequence set forth in SEQ ID NO: 1, the nucleotide sequence set forth in SEQ ID NO: 2, the nucleotide sequence set forth in SEQ ID NO: 23, and the SEQ ID NO: 24. Including oligonucleotides each having one or more base sequences selected from the base sequences described in
1 type or 2 types or more of said oligonucleotide of (b) is selected from the base sequence of sequence number: 3, the base sequence of sequence number: 4, and the base sequence of sequence number: 25 Including oligonucleotides each having a species or two or more nucleotide sequences,
The one or more oligonucleotides of (c) are the base sequence shown in SEQ ID NO: 5, the base sequence shown in SEQ ID NO: 6, the base sequence shown in SEQ ID NO: 26, and the SEQ ID NO: 27. Including oligonucleotides each having one or more base sequences selected from the base sequences described in
One or two or more oligonucleotides of (d) are the base sequence shown in SEQ ID NO: 7, the base sequence shown in SEQ ID NO: 8, the base sequence shown in SEQ ID NO: 28, and the sequence number 29. Including oligonucleotides each having one or more base sequences selected from the base sequences described in
The one or more oligonucleotides of (e) above are the base sequence shown in SEQ ID NO: 9, the base sequence shown in SEQ ID NO: 10, the base sequence shown in SEQ ID NO: 11, and the sequence number: 12 Comprising oligonucleotides each having one or more base sequences selected from the base sequence described in SEQ ID NO: 30, the base sequence set forth in SEQ ID NO: 30 and the base sequence set forth in SEQ ID NO: 31;
One or more oligonucleotides of the above (f) are the base sequence shown in SEQ ID NO: 13, the base sequence shown in SEQ ID NO: 14, the base sequence shown in SEQ ID NO: 32, and the SEQ ID NO: 33. Including oligonucleotides each having one or more base sequences selected from the base sequences described in
The one or more oligonucleotides of (g) are the nucleotide sequence set forth in SEQ ID NO: 15, the nucleotide sequence set forth in SEQ ID NO: 16 and the nucleotide sequence set forth in SEQ ID NO: 34, SEQ ID NO: 35. Comprising oligonucleotides each having one or more base sequences selected from the base sequence set forth in SEQ ID NO: 36, the base sequence set forth in SEQ ID NO: 36, and the base sequence set forth in SEQ ID NO: 37;
The one or more oligonucleotides of (h) are the nucleotide sequence set forth in SEQ ID NO: 17, the nucleotide sequence set forth in SEQ ID NO: 18, the nucleotide sequence set forth in SEQ ID NO: 38, and the sequence number: 39. Including oligonucleotides each having one or more base sequences selected from the base sequences described in
The one or more oligonucleotides of (i) above are the base sequence shown in SEQ ID NO: 19, the base sequence shown in SEQ ID NO: 20, the base sequence shown in SEQ ID NO: 40, the sequence number: 41. Including oligonucleotides each having one or more base sequences selected from the base sequences described in
The one or more oligonucleotides of (j) above are the base sequence set forth in SEQ ID NO: 21, the base sequence set forth in SEQ ID NO: 22, the base sequence set forth in SEQ ID NO: 42, and the SEQ ID NO: 43. The oligonucleotide which has 1 type, or 2 or more types of base sequences each selected from the base sequence as described in can be included.

  Furthermore, in the oligonucleotide set of the present invention, the oligonucleotide may be a primer. The oligonucleotide may be a probe.

According to the present invention, the following (A) to (J):
(A) One or two selected from the base sequence set forth in SEQ ID NO: 1, the base sequence set forth in SEQ ID NO: 2, the base sequence set forth in SEQ ID NO: 23, and the base sequence set forth in SEQ ID NO: 24 Oligonucleotide for detecting adenovirus each having a base sequence of at least species (B) From the base sequence set forth in SEQ ID NO: 3, the base sequence set forth in SEQ ID NO: 4 and the base sequence set forth in SEQ ID NO: 25 Oligonucleotide for detecting an enterovirus having one or more selected base sequences, respectively (C) The base sequence described in SEQ ID NO: 5, the base sequence described in SEQ ID NO: 6, and the sequence number: 26 For detecting herpes simplex virus type I each having one or more base sequences selected from the base sequence described in SEQ ID NO: 27 Gotonucleotide (D) One selected from the base sequence set forth in SEQ ID NO: 7, the base sequence set forth in SEQ ID NO: 8, the base sequence set forth in SEQ ID NO: 28, and the base sequence set forth in SEQ ID NO: 29 Or an oligonucleotide for detecting herpes simplex virus type II each having two or more types of base sequences (E) a base sequence set forth in SEQ ID NO: 9, a base sequence set forth in SEQ ID NO: 10, and a sequence set forth in SEQ ID NO: 11 Each having one or more base sequences selected from the base sequence described, the base sequence set forth in SEQ ID NO: 12, the base sequence set forth in SEQ ID NO: 30, and the base sequence set forth in SEQ ID NO: 31 Oligonucleotide for detecting group A rotavirus (F) Base sequence set forth in SEQ ID NO: 13, base sequence set forth in SEQ ID NO: 14, base sequence set forth in SEQ ID NO: 32 And an oligonucleotide for detecting a group C rotavirus each having one or more base sequences selected from the base sequences set forth in SEQ ID NO: 33 (G) the base sequence set forth in SEQ ID NO: 15, The base sequence set forth in SEQ ID NO: 16, the base sequence set forth in SEQ ID NO: 34, the base sequence set forth in SEQ ID NO: 35, the base sequence set forth in SEQ ID NO: 36, and the base sequence group set forth in SEQ ID NO: 37 (H) the nucleotide sequence described in SEQ ID NO: 17, the nucleotide sequence described in SEQ ID NO: 18, the nucleotide sequence described in SEQ ID NO: 18 : For detecting hepatitis A virus each having one or more base sequences selected from the base sequence set forth in 38 and the base sequence set forth in SEQ ID NO: 39 Oligonucleotide (I) One type selected from the base sequence set forth in SEQ ID NO: 19, the base sequence set forth in SEQ ID NO: 20, the base sequence set forth in SEQ ID NO: 40, and the base sequence set forth in SEQ ID NO: 41 Or an oligonucleotide for detecting a coronavirus each having two or more base sequences (J) the base sequence set forth in SEQ ID NO: 21, the base sequence set forth in SEQ ID NO: 22 and the base set forth in SEQ ID NO: 42 For detection of diarrhea-causing virus selected from the group consisting of oligonucleotides for detecting Aichi virus each having one or more base sequences selected from the sequence and the base sequence set forth in SEQ ID NO: 43 Oligonucleotides are provided.

  In the oligonucleotide of the present invention, the oligonucleotide may be a primer. The oligonucleotide may be a probe.

  According to the present invention, there is provided a solid phase carrier in which an oligonucleotide or an oligonucleotide contained in any of the oligonucleotide sets described above is immobilized on a solid support.

  Moreover, according to this invention, the detection kit for detecting the diarrhea origin virus containing the oligonucleotide set or oligonucleotide in any one of the said is also provided.

The present invention
Preparing a specimen sample that may contain nucleic acid derived from a diarrhea-causing virus;
PCR step of performing PCR using the nucleic acid as a template and the oligonucleotide set according to claim 7 or the oligonucleotide according to claim 11 as a primer;
A detection step for detecting a PCR product obtained by the PCR step;
The present invention relates to a method for detecting a diarrhea-causing virus.

  In the method for detecting a diarrhea-causing virus of the present invention, in the detection step, the oligonucleotide set or oligonucleotide of the present invention may be used as a probe when detecting the PCR product. The PCR may include a nested PCR.

The present invention is a method for diagnosing diarrhea,
Preparing a sample sample that may contain nucleic acid derived from a diarrhea-causing virus from a test sample collected from an animal; and
PCR step of performing PCR using the nucleic acid as a template and the oligonucleotide set according to claim 7 or the oligonucleotide according to claim 11 as a primer;
A detection step for detecting a PCR product obtained by the PCR step;
A diagnostic step of diagnosing either the risk of developing the diarrhea of the animal based on the detection result of the detection step, whether the animal is suffering from diarrhea and the degree of progression of the diarrhea;
The present invention relates to a method for diagnosing diarrhea.

  In the method for diagnosing diarrhea of the present invention, the detection step may use the oligonucleotide set or oligonucleotide of the present invention as a probe when detecting the PCR product. The PCR may include a nested PCR.

  The oligonucleotide set for detecting diarrhea-causing virus of the present invention includes adenovirus hexon gene, enterovirus 5 'untranslated region, herpes simplex virus type I DNA polymerase gene, herpes simplex virus type II glycoprotein gene VP4 gene of group A rotavirus, VP4 gene of group C rotavirus, protease gene of astrovirus, 5 'untranslated region of hepatitis A virus, surface glycoprotein gene of coronavirus and VP1 gene of Aichi virus And an oligonucleotide that hybridizes with a polynucleotide encoding each of two or more regions or a part thereof.

  According to the present invention, two or more types of diarrhea-causing viruses can be detected by targeting a specific site of each virus. In particular, when the oligonucleotide set of the present invention is used as a PCR primer, each primer pair amplifies a specific region of the genome of the diarrhea-causing virus that is specifically targeted, so Viruses can be detected quickly and exhaustively.

  Hereinafter, an oligonucleotide set for detecting a diarrhea-causing virus that is an embodiment of the present invention will be described, an oligonucleotide constituting the oligonucleotide set, a solid-phase carrier containing the oligonucleotide set or oligonucleotide, A detection kit for detecting a diarrhea-causing virus, a method for detecting a diarrhea-causing virus, and a method for diagnosing diarrhea are sequentially described.

(Analysis target)
The object of analysis by the oligonucleotide set and oligonucleotide of the present invention are adenovirus, enterovirus, herpes simplex virus type I, herpes simplex virus type II, group A rotavirus, group C rotavirus, astrovirus, hepatitis A virus, corona Virus and Aichi virus, and more specifically, DNA or RNA of these viruses. Further, the analysis target may be one type or a plurality of types among these ten types of viruses, but it is preferable that a plurality of types are detected. Specifically, it is preferable to detect 5 or more types of viruses, and it is more preferable to detect 8 or more types of viruses. Moreover, when detecting multiple types of viruses, it is preferable that these are detected simultaneously.

(Adenovirus analysis target)
Examples of the viral DNA or RNA (target) to be analyzed include the following. That is, the adenovirus is preferably a hexon gene, more preferably the base sequence set forth in SEQ ID NO: 1, the base sequence set forth in SEQ ID NO: 2, and the sequence set forth in SEQ ID NO: 23 in the hexon gene of the adenovirus. It is a region sandwiched by any two base sequences of the base sequence, the base sequence set forth in SEQ ID NO: 24, and their complementary sequences. In addition, similar sequences of these specific base sequences possessed by the same virus can also be targeted. As such a target sequence, for example, one or several base substitutions, deletions, insertions and additions, or two or more kinds of modifications are made to the base sequence described in any one of SEQ ID NOs: 1, 2, 23 and 24 Examples include a region sandwiched by any two of the base sequences. Such a sequence can be appropriately searched using, for example, known software.

  Here, the nucleotide sequence shown in SEQ ID NO: 1, its complementary sequence, or a similar sequence thereof, specifically, the 2471st to 2487th positions in the adenovirus type 7a hexon gene (GenBank Accession No: AF065066) , The 2480th to 2496th nucleotide sequences in the adenovirus type 7 hexon gene (GenBank Accession No: AF065065), and the adenovirus type 41 hexon gene (GenBank Accession No: D13781) from the 2525th position to the 25th position. 2541st nucleotide sequence, 20151st to 20167th nucleotide sequence in adenovirus type 9 hexon gene (GenBank Accession No: AJ854486), 20630th position in adenovirus type 11 hexon gene (GenBank Accession No: AY163756) To 20646th base sequence, adenovirus type F Hexon gene (GenBank Accession No: L19443) from the 19944-th first 19,960 th nucleotide sequence are exemplified in.

  Further, the base sequence described in SEQ ID NO: 2, its complementary sequence, or a similar sequence thereof, specifically, the 2873th to 2889th in the adenovirus type 7a hexon gene (GenBank Accession No: AF065066) The nucleotide sequence, the 2882th to 2898th nucleotide sequences in the adenovirus type 7 hexon gene (GenBank Accession No: AF065065), the 2927th to 2943th in the adenovirus type 41 hexon gene (GenBank Accession No: D13781) The 20th base sequence, the 2053rd to 2069th base sequence in the adenovirus type 9 hexon gene (GenBank Accession No: AJ854486), the 210th position in the adenovirus type 11 hexon gene (GenBank Accession No: AY163756) The 21048th nucleotide sequence, adenovirus type F ROCEPHIN gene (GenBank Accession No: L19443) from the 20362 th second 20346 th nucleotide sequence are exemplified in.

  The nucleotide sequence shown in SEQ ID NO: 23, its complementary sequence, or a similar sequence thereof, specifically, the 2690th to 2709th positions in the adenovirus type 7a hexon gene (GenBank Accession No: AF065066) The nucleotide sequence, the 2699th to 2718th nucleotide sequences in the adenovirus type 7 hexon gene (GenBank Accession No: AF065065), and the 2744th to 2766th in the adenovirus type 41 hexon gene (GenBank Accession No: D13781). The 20th base sequence, the 20370th to 20389th base sequences in the adenovirus type 9 hexon gene (GenBank Accession No: AJ854486), the 20th position in the adenovirus type 11 hexon gene (GenBank Accession No: AY163756) The 20868th nucleotide sequence, adenovirus type F Hexon gene (GenBank Accession No: L19443) from the 20163 th second 20182 th nucleotide sequence are exemplified in.

  Further, the base sequence shown in SEQ ID NO: 24, its complementary sequence, or a similar sequence thereof, specifically, the 2840th to 2859th positions in the adenovirus type 7a hexon gene (GenBank Accession No: AF065066) Base sequence, 2849th to 2868th base sequence in adenovirus type 7 hexon gene (GenBank Accession No: AF065065), 2894th to 2913 in adenovirus type 41 hexon gene (GenBank Accession No: D13781) The 20th base sequence from the 20520th to 20539th base sequence in the adenovirus type 9 hexon gene (GenBank Accession No: AJ854486), the 20th base sequence from the adenovirus type 11 hexon gene (GenBank Accession No: AY163756) 21018th nucleotide sequence, adenovirus type F Hexon gene (GenBank Accession No: L19443) from the 20313 th second 20332 th nucleotide sequence are exemplified in.

(Analysis target of enterovirus)
The enterovirus is preferably the 5 ′ untranslated region of the enterovirus, more preferably the base sequence set forth in SEQ ID NO: 3 in the 5 ′ untranslated region of the enterovirus, the base sequence set forth in SEQ ID NO: 4, and the sequence number: 25 is a region sandwiched between any two base sequences of the base sequence described in 25 and their complementary sequences. In addition, similar sequences of these specific base sequences possessed by the same virus can also be targeted. As such a target sequence, for example, one or several base substitutions, deletions, insertions and additions, or two or more types thereof have been modified in the base sequence described in any of SEQ ID NOs: 3, 4 and 25 Examples include a region sandwiched between any two of the base sequences.

  Here, as the base sequence described in SEQ ID NO: 3, its complementary sequence, or a similar sequence thereof, specifically, from the 162nd to the 182nd in the untranslated region of poliovirus type 1 (GenBank Accession No: AY184219) The second base sequence is mentioned. In addition, as the base sequence described in SEQ ID NO: 4, its complementary sequence, or a similar sequence thereof, specifically, the 531st to 552th positions in the untranslated region of poliovirus type 1 (GenBank Accession No: AY184219) The nucleotide sequence of Further, as the base sequence shown in SEQ ID NO: 25, its complementary sequence or its similar sequence, specifically, the 357th to 375th positions in the untranslated region of poliovirus type 1 (GenBank Accession No: AY184219) And the 361st to 379th nucleotide sequences in the Coxsackievirus B5 untranslated region (GenBank Accession No: AF114383).

(Analysis target of herpes simplex virus type I)
The herpes simplex virus type I is preferably a DNA polymerase gene, more preferably the base sequence set forth in SEQ ID NO: 5 in the herpes simplex virus type I DNA polymerase gene, the base sequence set forth in SEQ ID NO: 6, and the sequence It is a region sandwiched by any two base sequences of the base sequence described in No. 26, the base sequence described in SEQ ID No. 27, and their complementary sequences. In addition, similar sequences of these specific base sequences possessed by the same virus can also be targeted. As such a target sequence, for example, one or several base substitutions, deletions, insertions and additions or two or more kinds of modifications are made to the base sequence described in any of SEQ ID NOs: 5, 6, 26 and 27 Examples include a region sandwiched by any two of the base sequences.

  Here, as the base sequence described in SEQ ID NO: 5, its complementary sequence, or a similar sequence, specifically, from the 64364th position in the herpes simplex virus type I DNA polymerase gene (GenBank Accession No: X14112) The 64383rd base sequence is mentioned. Further, the base sequence described in SEQ ID NO: 6, its complementary sequence, or a similar sequence thereof, specifically, the 64812th to 64829th in the herpes simplex virus type I DNA polymerase gene (GenBank Accession No: X14112) The second base sequence is mentioned. In addition, the base sequence described in SEQ ID NO: 26, its complementary sequence, or a similar sequence thereof, specifically, from the 64482th to the 64501st in the herpes simplex virus type I DNA polymerase gene (GenBank Accession No: X14112) The nucleotide sequence of Further, the base sequence described in SEQ ID NO: 27, its complementary sequence, or a similar sequence thereof, specifically, from the 64716th to the 64735 in the herpes simplex virus type I DNA polymerase gene (GenBank Accession No: X14112) The second base sequence is mentioned.

(Analysis subject of herpes simplex virus type II)
Herpes simplex virus type II is preferably a glycoprotein gene, more preferably the base sequence set forth in SEQ ID NO: 7 in the herpes simplex virus type II glycoprotein gene, the base sequence set forth in SEQ ID NO: 8, sequence It is a region sandwiched by any two base sequences of the base sequence described in No. 28, the base sequence described in SEQ ID No. 29, and their complementary sequences. In addition, similar sequences of these specific base sequences possessed by the same virus can also be targeted. Examples of such a target sequence include one or several base substitutions, deletions, insertions and additions, or two or more kinds of modifications to the base sequence described in any of SEQ ID NOs: 7, 8, 28 and 29. Examples include a region sandwiched by any two of the base sequences.

  Here, as the base sequence described in SEQ ID NO: 6, its complementary sequence, or a similar sequence thereof, specifically, from the 54561st position in the herpes simplex virus type II glycoprotein gene (GenBank Accession No: Z86099) The 54580th base sequence is mentioned. Further, the base sequence described in SEQ ID NO: 7, its complementary sequence, or a similar sequence thereof, specifically, the 54877th to 54858 in the herpes simplex virus type II glycoprotein gene (GenBank Accession No: Z86099) The second base sequence is mentioned. Further, the base sequence described in SEQ ID NO: 28, its complementary sequence, or a similar sequence thereof, specifically, the 54682 to 54701 in the herpes simplex virus type II glycoprotein gene (GenBank Accession No: Z86099) The second base sequence is mentioned. Further, the base sequence described in SEQ ID NO: 29, its complementary sequence, or a similar sequence thereof, specifically, the 54854th to 54835 in the herpes simplex virus type II glycoprotein gene (GenBank Accession No: Z86099) The second base sequence is mentioned.

(Analysis target of group A rotavirus)
The group A rotavirus is preferably the VP4 gene, more preferably the base sequence set forth in SEQ ID NO: 9 in the VP4 gene of the group A rotavirus, the base sequence set forth in SEQ ID NO: 10, and the sequence set forth in SEQ ID NO: 11. It is sandwiched between any two nucleotide sequences among the nucleotide sequence described, the nucleotide sequence set forth in SEQ ID NO: 12, the nucleotide sequence set forth in SEQ ID NO: 30, the nucleotide sequence set forth in SEQ ID NO: 31 and their complementary sequences It is an area. In addition, similar sequences of these specific base sequences possessed by the same virus can also be targeted. Examples of such a target sequence include any one of substitution, deletion, insertion and addition of one or several bases to the base sequence described in any of SEQ ID NOs: 9, 10, 11, 12, 30 and 31 or Examples include a region sandwiched by any two of the two or more types of modified base sequences.

  Here, as the base sequence described in SEQ ID NO: 9, its complementary sequence, or a similar sequence thereof, specifically, the 33rd to 56th in the VP4 gene (GenBank Accession No: AB008279) of group A rotavirus YO. The second base sequence is mentioned. Further, the base sequence described in SEQ ID NO: 10, its complementary sequence, or a similar sequence thereof, specifically, the 418th to 441th positions in the VP4 gene (GenBank Accession No: AB008279) of group A rotavirus YO The nucleotide sequence of Further, as the base sequence described in SEQ ID NO: 11, its complementary sequence, or a similar sequence thereof, specifically, the 33rd to 56th in the VP4 gene (GenBank Accession No: M60600) of group A rotavirus 69M And the 33rd to 56th nucleotide sequences in the VP4 gene (GenBank Accession No: D16346) of group A rotavirus SA11. Further, the base sequence described in SEQ ID NO: 12, its complementary sequence, or a similar sequence thereof is specifically the 421st to 444th in the VP4 gene (GenBank Accession No: M60600) of group A rotavirus 69M. And the 421st to 444th nucleotide sequences in the VP4 gene (GenBank Accession No: D16346) of group A rotavirus 69MSA11.

  The nucleotide sequence shown in SEQ ID NO: 30, its complementary sequence or its similar sequence is specifically the 67th to the 89th in the VP4 gene (GenBank Accession No: AB008279) of group A rotavirus YO. Of the VP4 gene (GenBank Accession No: AF401755) of Group A rotavirus KO-2, and the VP4 gene (GenBank Accession No: L33895) of Group A rotavirus ST3. Examples include the 67th to 89th nucleotide sequences. The nucleotide sequence shown in SEQ ID NO: 31, its complementary sequence, or a similar sequence thereof is specifically the 67th to 89th positions in the VP4 gene (GenBank Accession No: M60600) of group A rotavirus 69M. And the 67th to 89th nucleotide sequences in the VP4 gene (GenBank Accession No: D16346) of group A rotavirus SA11.

(Analysis subject of group C rotavirus)
The group C rotavirus is preferably a VP4 gene, more preferably the base sequence set forth in SEQ ID NO: 13 in the VP4 gene of group C rotavirus, the base sequence set forth in SEQ ID NO: 14, and the sequence set forth in SEQ ID NO: 32. It is a region sandwiched between any two base sequences of the base sequence described, the base sequence set forth in SEQ ID NO: 33, and their complementary sequences. In addition, similar sequences of these specific base sequences possessed by the same virus can also be targeted. Examples of such a target sequence include one or several base substitutions, deletions, insertions and additions, or two or more types of modifications to the base sequence described in any one of SEQ ID NOs: 13, 14, 32 and 33. Examples include a region sandwiched by any two of the base sequences.

  Here, as the base sequence described in SEQ ID NO: 13, its complementary sequence, or a similar sequence thereof, specifically, the 11th to the 30th in the VP4 gene (GenBank Accession No: AF323981) of group C rotavirus The nucleotide sequence of Further, as the base sequence described in SEQ ID NO: 14, its complementary sequence, or a similar sequence thereof, specifically, the 317th to 337th positions in the VP4 gene (GenBank Accession No: AF323981) of group C rotavirus Examples include base sequences. Further, as the base sequence described in SEQ ID NO: 32, its complementary sequence or its similar sequence, specifically, the 31st to 52nd in the VP4 gene (GenBank Accession No: AF323981) of group C rotavirus Examples include base sequences. In addition, as the base sequence described in SEQ ID NO: 33, its complementary sequence, or a similar sequence thereof, specifically, the 229th to 250th positions in the VP4 gene (GenBank Accession No: AF323981) of group C rotavirus Examples include base sequences.

(Astrovirus analysis target)
The astrovirus is preferably a protease gene, more preferably the base sequence set forth in SEQ ID NO: 15 in the protease gene of the astrovirus, the base sequence set forth in SEQ ID NO: 16, and the base sequence set forth in SEQ ID NO: 34. These are regions sandwiched by any two base sequences of the base sequence set forth in SEQ ID NO: 35, the base sequence set forth in SEQ ID NO: 36, the base sequence set forth in SEQ ID NO: 37, and their complementary sequences. In addition, similar sequences of these specific base sequences possessed by the same virus can also be targeted. Examples of such a target sequence include any one of substitution, deletion, insertion and addition of one or several bases in the base sequence described in any one of SEQ ID NOs: 15, 16, 34, 35, 36 and 37, or Examples include a region sandwiched by any two of the two or more types of modified base sequences.

  Here, as the base sequence described in SEQ ID NO: 15, its complementary sequence, or a similar sequence thereof, specifically, the 130th to 149th positions in the Astrovirus type 2 protease gene (GenBank Accession No: L13745) The nucleotide sequence of Further, the base sequence described in SEQ ID NO: 16, its complementary sequence, or a similar sequence thereof, specifically, the 436th to 455th positions in the Astrovirus type 2 protease gene (GenBank Accession No: L13745) Examples include base sequences. Further, as the base sequence shown in SEQ ID NO: 34, its complementary sequence, or a similar sequence thereof, specifically, the 170th to 191st in the Astrovirus type 2 protease gene (GenBank Accession No: L13745) Examples include base sequences. In addition, as the base sequence described in SEQ ID NO: 35, its complementary sequence, or a similar sequence thereof, specifically, from the 400th to the 421st in the Astrovirus type 2 protease gene (GenBank Accession No: L13745) This corresponds to the base sequence. Further, as the base sequence described in SEQ ID NO: 36, its complementary sequence or a similar sequence thereof, specifically, the 41st to 62nd base sequences in the Astrovirus type 6 protease gene or Astrovirus type 7 The 41st to 62nd base sequences of the protease gene are mentioned. In addition, as the base sequence described in SEQ ID NO: 37, its complementary sequence, or a similar sequence thereof, specifically, the 287th to 309th base sequences in the Astrovirus type 6 protease gene or Astrovirus type 7 And the 287th to 309th nucleotide sequences in the protease gene.

(Target for analysis of hepatitis A virus)
For the hepatitis A virus, preferably a 5 ′ untranslated region, more preferably the base sequence set forth in SEQ ID NO: 17 and the base sequence set forth in SEQ ID NO: 18 in the 5 ′ untranslated region of hepatitis A virus A region sandwiched between any two base sequences of the base sequence set forth in SEQ ID NO: 38, the base sequence set forth in SEQ ID NO: 39, and their complementary sequences. In addition, similar sequences of these specific base sequences possessed by the same virus can also be targeted. As such a target sequence, for example, one or several base substitutions, deletions, insertions and additions, or two or more kinds of modifications are made to the base sequence described in any one of SEQ ID NOs: 17, 18, 38 and 39 Examples include a region sandwiched by any two of the base sequences.

  Here, as the base sequence described in SEQ ID NO: 17, its complementary sequence, or a similar sequence thereof, specifically, the 221nd in the 5 ′ untranslated region (GenBank Accession No: AB020569) of hepatitis A virus FH3 To the 240th nucleotide sequence. Further, the base sequence described in SEQ ID NO: 18, its complementary sequence, or a similar sequence thereof, specifically, from the 603rd in the 5 ′ untranslated region (GenBank Accession No: AB020569) of hepatitis A virus FH3 Examples include the 622nd nucleotide sequence and the 606th to 625th nucleotide sequences in the 5 ′ untranslated region (GenBank Accession No: AF314208) of hepatitis A virus. Further, the base sequence described in SEQ ID NO: 38, its complementary sequence, or a similar sequence thereof, specifically, from the 391st in the 5 ′ untranslated region (GenBank Accession No: AB020569) of hepatitis A virus FH3 Examples include the 411st nucleotide sequence and the 394th to 414th nucleotide sequences in the 5 'untranslated region (GenBank Accession No: AF314208) of hepatitis A virus. Further, the base sequence described in SEQ ID NO: 39, its complementary sequence, or a similar sequence thereof, specifically, from the 576th position in the 5 ′ untranslated region (GenBank Accession No: AB020569) of hepatitis A virus FH3 Examples include the 595th base sequence.

(Coronavirus analysis target)
The coronavirus is preferably a surface glycoprotein gene, more preferably the coronavirus surface glycoprotein gene, the nucleotide sequence set forth in SEQ ID NO: 19; the nucleotide sequence set forth in SEQ ID NO: 20; A region sandwiched by any two base sequences of the base sequence described, the base sequence set forth in SEQ ID NO: 41, and their complementary sequences. In addition, similar sequences of these specific base sequences possessed by the same virus can also be targeted. As such a target sequence, for example, one or several base substitutions, deletions, insertions and additions, or two or more kinds of modifications are made to the base sequence described in any one of SEQ ID NOs: 19, 20, 40 and 41 Examples include a region sandwiched by any two of the base sequences.

  Here, as the base sequence described in SEQ ID NO: 19, its complementary sequence or a similar sequence, specifically, from the 235th to the 254th in the surface glycoprotein gene of Coronavirus OC43 (GenBank Accession No: S62886) The second base sequence is mentioned. The nucleotide sequence shown in SEQ ID NO: 20, its complementary sequence, or a similar sequence thereof, specifically, the 574th to the 593rd in the coronavirus OC43 surface glycoprotein gene (GenBank Accession No: S62886) The nucleotide sequence of Further, the base sequence described in SEQ ID NO: 40, its complementary sequence, or a similar sequence thereof, specifically, the 268th to 289th positions in the coronavirus OC43 surface glycoprotein gene (GenBank Accession No: S62886) The nucleotide sequence of Further, as the base sequence described in SEQ ID NO: 41, its complementary sequence, or a similar sequence, specifically, the 504th to 525th in the surface glycoprotein gene (GenBank Accession No: S62886) of coronavirus OC43 The nucleotide sequence of

(Analysis target of Aichi virus)
For Aichi virus, preferably the VP1 gene, more preferably the nucleotide sequence set forth in SEQ ID NO: 21 in the VP1 gene of Aichi virus, the nucleotide sequence set forth in SEQ ID NO: 22 and the nucleotide sequence set forth in SEQ ID NO: 42 A region sandwiched by any two base sequences of the base sequence set forth in SEQ ID NO: 43 and their complementary sequences. In addition, similar sequences of these specific base sequences possessed by the same virus can also be targeted. Examples of such a target sequence include one or several base substitutions, deletions, insertions and additions, or two or more kinds of modifications to the base sequence described in any one of SEQ ID NOs: 21, 22, 42 and 43. Examples include a region sandwiched by any two of the base sequences.

  Here, as the base sequence described in SEQ ID NO: 21, its complementary sequence, or a similar sequence thereof, specifically, the 3362th to 3379th bases in the VP1 gene (GenBank Accession No: NC_001918) of Aichivirus Examples include sequences. In addition, as the base sequence described in SEQ ID NO: 22, its complementary sequence, or a similar sequence thereof, specifically, the base sequence from the 3739th position to the 3756th position in the VP1 gene (GenBank Accession No: NC — 001918) of Aichivirus Is mentioned. In addition, as the base sequence described in SEQ ID NO: 42, its complementary sequence, or a similar sequence thereof, specifically, the base sequence from the 3551st position to the 3573th base position in the VP1 gene (GenBank Accession No: NC — 001918) of Aichi virus Is mentioned. In addition, as the base sequence described in SEQ ID NO: 43, its complementary sequence, or a similar sequence thereof, specifically, the 3736th to 3756th base sequences in the VP1 gene (GenBank Accession No: NC — 001918) of Aichivirus Is mentioned.

(Oligonucleotide set and oligonucleotide)
The oligonucleotide set of the present invention includes an oligonucleotide for detecting the target. Examples of such oligonucleotides include primers used for PCR and the like, probes used for hybridization by various methods, and the like. When these oligonucleotides are used as primers, even if multiple types of primer pairs with different analysis targets are included, each primer pair amplifies a specific region of the genome of the virus that causes diarrhea that is specifically targeted. Therefore, it is possible to quickly and comprehensively detect two or more types of diarrhea-causing viruses. In addition, since the oligonucleotide for detecting such a target is only required to be able to detect the target, it may be a PCR primer that amplifies a wide range including the target, or hybridizes with all or part of the target. It may be a probe. The design of primers and probes can also be performed by software available through the Internet or disclosed software, respectively.

  Further, when the oligonucleotide set of the present invention is provided as, for example, a detection kit, each oligonucleotide may be provided in an individual state, or in a state where two or more kinds of oligonucleotides are mixed. It may be provided.

  When the oligonucleotide set or oligonucleotide of the present invention is used as a probe, the oligonucleotide is preferably immobilized on a solid support. Such a solid support may be appropriately selected depending on the application, and examples thereof include a slide glass and a plastic plate. The probe may be appropriately labeled. Examples of substances used for labeling generally include fluorescent substances and biotin.

(Adenonucleotide set or oligonucleotide for adenovirus detection)
Examples of such oligonucleotides include primers and probes having the base sequences set forth in SEQ ID NOs: 1, 2, 23 and 24 for the above target of the adenovirus hexon gene. When these oligonucleotides are used as primers, the oligonucleotide described in SEQ ID NO: 1 is used as a sense primer (primer on the 5 ′ end side), and the oligonucleotide described in SEQ ID NO: 2 is used as an antisense primer (3 ′ end side). 419 bases corresponding to the region sandwiched between both primers in the adenovirus hexon gene can be amplified. Moreover, after performing PCR or RT-PCR using the oligonucleotide described in SEQ ID NO: 1 and the oligonucleotide described in SEQ ID NO: 2, the oligonucleotide described in SEQ ID NO: 23 with respect to the PCR product Nested PCR can also be performed using as a sense primer and the oligonucleotide set forth in SEQ ID NO: 24 as an antisense primer. Thereby, 170 bases corresponding to the region sandwiched between both primers in the hexon gene of the adenovirus gene can be amplified. Alternatively, for the PCR product obtained by combining the oligonucleotide shown in SEQ ID NO: 1 and the oligonucleotide shown in SEQ ID NO: 2, the oligonucleotide shown in SEQ ID NO: 1 and the oligonucleotide shown in SEQ ID NO: 24 are used. Semi-nested PCR can also be performed by a combination with a nucleotide or a combination of the oligonucleotide described in SEQ ID NO: 2 and the oligonucleotide described in SEQ ID NO: 23. In addition, an oligonucleotide having the base sequence described in any of SEQ ID NOs: 1, 2, 23 and 24 can be used as a probe for various hybridizations such as microarrays and electrophoresis.

(An oligonucleotide set or oligonucleotide for detecting enterovirus)
Moreover, with respect to the above target of the 5 ′ untranslated region of enterovirus, primers and probes each having the base sequence described in SEQ ID NOs: 3, 4 and 25 can be mentioned. When these oligonucleotides are used as primers, PCR or RT-PCR is performed using the oligonucleotide shown in SEQ ID NO: 3 as a sense primer and the oligonucleotide shown in SEQ ID NO: 4 as an antisense primer. Of the untranslated region, 383 bases corresponding to the region sandwiched between both primers can be amplified. In addition, after performing PCR using the oligonucleotide shown in SEQ ID NO: 3 and the oligonucleotide shown in SEQ ID NO: 4, the oligonucleotide shown in SEQ ID NO: 25 was used as a sense primer for the PCR product. Seminested PCR can also be performed using the oligonucleotide described in SEQ ID NO: 4 as an antisense primer. In addition, the oligonucleotide having the base sequence described in any of SEQ ID NOs: 3, 4 and 25 can be used as a probe for various hybridizations such as microarrays and electrophoresis.

(Oligonucleotide set or oligonucleotide for detection of herpes simplex virus type I)
Examples of the target of the herpes simplex virus type I DNA polymerase gene include primers and probes each having the nucleotide sequence set forth in SEQ ID NOs: 5, 6, 26 and 27. When these oligonucleotides are used as primers, PCR or RT-PCR is performed using the oligonucleotide shown in SEQ ID NO: 5 as a sense primer and the oligonucleotide shown in SEQ ID NO: 6 as an antisense primer. It is possible to amplify 468 bases corresponding to the region sandwiched between both primers in the virus type I DNA polymerase gene. In addition, after performing PCR or RT-PCR using the oligonucleotide described in SEQ ID NO: 5 and the oligonucleotide described in SEQ ID NO: 6, the oligonucleotide described in SEQ ID NO: 26 is added to the PCR product. Nested PCR can also be performed using the sense primer and the oligonucleotide set forth in SEQ ID NO: 27 as an antisense primer. Thereby, 252 bases corresponding to the region sandwiched between both primers in the herpes simplex virus type I DNA polymerase gene can be amplified. Alternatively, for the PCR product obtained by combining the oligonucleotide shown in SEQ ID NO: 5 and the oligonucleotide shown in SEQ ID NO: 6, the oligonucleotide shown in SEQ ID NO: 5 and the oligo shown in SEQ ID NO: 27 Semi-nested PCR can also be performed by a combination with a nucleotide or a combination of the oligonucleotide described in SEQ ID NO: 6 and the oligonucleotide described in SEQ ID NO: 26. In addition, the oligonucleotide having the base sequence described in any one of SEQ ID NOs: 5, 6, 26 and 27 can be used as a probe for various hybridizations such as microarrays and electrophoresis.

(Oligonucleotide set or oligonucleotide for detection of herpes simplex virus type II)
For the above target of the herpes simplex virus type II glycoprotein gene, primers and probes having the nucleotide sequences set forth in SEQ ID NOs: 7, 8, 28 and 29, respectively, can be mentioned. When these oligonucleotides are used as primers, PCR or RT-PCR is performed using the oligonucleotide shown in SEQ ID NO: 7 as a sense primer and the oligonucleotide shown in SEQ ID NO: 8 as an antisense primer. It is possible to amplify 317 bases corresponding to the region sandwiched between both primers in the virus type II glycoprotein gene. In addition, after performing PCR or RT-PCR using the oligonucleotide described in SEQ ID NO: 7 and the oligonucleotide described in SEQ ID NO: 8, the PCR product is subjected to the oligonucleotide described in SEQ ID NO: 28. Nested PCR can also be performed using as a sense primer and the oligonucleotide set forth in SEQ ID NO: 29 as an antisense primer. Thereby, 173 bases corresponding to the region sandwiched between both primers in the herpes simplex virus type II glycoprotein gene can be amplified. Alternatively, for the PCR product obtained by combining the oligonucleotide described in SEQ ID NO: 7 and the oligonucleotide described in SEQ ID NO: 8, the oligonucleotide described in SEQ ID NO: 7 and the oligo described in SEQ ID NO: 29 Semi-nested PCR can also be performed by a combination with a nucleotide or a combination of the oligonucleotide shown in SEQ ID NO: 8 and the oligonucleotide shown in SEQ ID NO: 28. In addition, the oligonucleotide having the base sequence described in any of SEQ ID NOs: 7, 8, 28 and 29 can be used as a probe for various hybridizations such as microarrays and electrophoresis.

(Oligonucleotide set or oligonucleotide for group A rotavirus detection)
For the above target of the VP4 gene of group A rotavirus, primers and probes each having the base sequence set forth in SEQ ID NOs: 9-12, 30, and 31 can be mentioned. When these oligonucleotides are used as primers, PCR or RT-PCR is carried out using the oligonucleotide shown in SEQ ID NO: 9 as a sense primer and the nucleotide shown in SEQ ID NO: 10 as an antisense primer. It is possible to amplify 409 bases corresponding to the region between both primers in the viral VP4 gene. Alternatively, by performing PCR or RT-PCR using the oligonucleotide shown in SEQ ID NO: 11 as a sense primer and the nucleotide shown in SEQ ID NO: 12 as an antisense primer, both primers of the VP4 gene of group A rotavirus 412 bases corresponding to the region sandwiched between can be amplified. In addition, when performing PCR or RT-PCR using these oligonucleotides, a mixed primer obtained by mixing the oligonucleotides described in SEQ ID NOs: 9 to 12 may be used.

  In addition, after PCR or RT-PCR was performed using the combination of oligonucleotides described in SEQ ID NOs: 9 and 10 or the combination of oligonucleotides described in SEQ ID NOs: 11 and 12, : A semi-nested PCR using the oligonucleotide described in 30 as a sense primer and the nucleotide set forth in SEQ ID NO: 10 as an antisense primer allows the region VP4 of group A rotavirus to be sandwiched between both primers. The corresponding 375 bases can be amplified. Alternatively, by performing semi-nested PCR using the oligonucleotide set forth in SEQ ID NO: 31 as a sense primer and the nucleotide set forth in SEQ ID NO: 12 as an antisense primer, it is sandwiched between both primers of the VP4 gene of Group A rotavirus. 378 bases corresponding to the region can be amplified. When semi-nested PCR is performed using these oligonucleotides, a mixed primer obtained by mixing the oligonucleotides described in SEQ ID NOs: 10, 12, 30, and 31 may be used. Moreover, the oligonucleotide which has the base sequence in any one of sequence number: 9-12,30 and 31 can also be used as various hybridization probes, such as a microarray and electrophoresis.

(Oligonucleotide set or oligonucleotide for group C rotavirus detection)
Examples of the target of the VP4 gene of group C rotavirus include primers and probes each having the base sequence set forth in SEQ ID NOs: 13, 14, 32 and 33. When these oligonucleotides are used as primers, PCR or RT-PCR is carried out using the oligonucleotide set forth in SEQ ID NO: 13 as a sense primer and the oligonucleotide set forth in SEQ ID NO: 14 as an antisense primer. 327 bases corresponding to the region sandwiched between both primers in the rotavirus VP4 gene can be amplified. In addition, after performing PCR or RT-PCR using the oligonucleotide shown in SEQ ID NO: 13 and the oligonucleotide shown in SEQ ID NO: 14, the oligonucleotide shown in SEQ ID NO: 32 is obtained with respect to the PCR product. Nested PCR can also be performed using as a sense primer and the oligonucleotide set forth in SEQ ID NO: 33 as an antisense primer. As a result, 159 bases corresponding to the region sandwiched between both primers in the VP4 gene of group C rotavirus can be amplified. Alternatively, for the PCR product obtained by combining the oligonucleotide shown in SEQ ID NO: 13 and the oligonucleotide shown in SEQ ID NO: 14, the oligonucleotide shown in SEQ ID NO: 13 and the oligonucleotide shown in SEQ ID NO: 33 Semi-nested PCR can also be performed by a combination with a nucleotide or a combination of the oligonucleotide described in SEQ ID NO: 14 and the oligonucleotide described in SEQ ID NO: 32. Moreover, the oligonucleotide which has the base sequence in any one of sequence number: 13, 14, 32 and 33 can also be used as a probe of various hybridizations, such as a microarray and electrophoresis.

(An oligonucleotide set or oligonucleotide for detecting astrovirus)
Examples of the target of the astrovirus protease gene include primers and probes each having the base sequence set forth in SEQ ID NOs: 15, 16, and 34 to 37. When these oligonucleotides are used as primers, by performing PCR or RT-PCR using the oligonucleotide set forth in SEQ ID NO: 15 as a sense primer and the oligonucleotide set forth in SEQ ID NO: 16 as an antisense primer, Astrovirus 326 bases corresponding to the region sandwiched between both primers can be amplified. Moreover, after performing PCR or RT-PCR using the oligonucleotide described in SEQ ID NO: 15 and the oligonucleotide described in SEQ ID NO: 16, the oligonucleotide described in SEQ ID NO: 34 with respect to the PCR product And a combination of the oligonucleotide described in SEQ ID NO: 35 or the combination of the oligonucleotide described in SEQ ID NO: 36 and the oligonucleotide described in SEQ ID NO: 37 can also be used for semi-nested PCR. Thus, 252 bases corresponding to the region sandwiched between both primers can be amplified in the former case of the astrovirus protease gene, and 269 residues can be amplified in the latter case. Moreover, the oligonucleotide which has the base sequence in any one of sequence number: 15,16 and 34-37 can also be used as a probe of various hybridizations, such as a microarray and electrophoresis.

(Oligonucleotide set or oligonucleotide for detection of hepatitis A virus)
For the above target of the 5 ′ untranslated region of hepatitis A virus, there may be mentioned primers and probes having the nucleotide sequences set forth in SEQ ID NOs: 13, 14, 32 and 33, respectively. When these oligonucleotides are used as primers, PCR or RT-PCR is performed using the oligonucleotide set forth in SEQ ID NO: 13 as a sense primer and the oligonucleotide set forth in SEQ ID NO: 14 as an antisense primer. It is possible to amplify 407 bases corresponding to the region sandwiched between both primers in the 5 ′ untranslated region of hepatitis virus. In addition, after performing PCR or RT-PCR using the oligonucleotide shown in SEQ ID NO: 13 and the oligonucleotide shown in SEQ ID NO: 14, the oligonucleotide shown in SEQ ID NO: 32 is obtained with respect to the PCR product. Nested PCR can also be performed using as a sense primer and the oligonucleotide set forth in SEQ ID NO: 33 as an antisense primer. Thereby, 207 bases corresponding to the region sandwiched between both primers in the 5 ′ untranslated region of hepatitis A virus can be amplified. Alternatively, for the PCR product obtained by combining the oligonucleotide shown in SEQ ID NO: 13 and the oligonucleotide shown in SEQ ID NO: 14, the oligonucleotide shown in SEQ ID NO: 13 and the oligonucleotide shown in SEQ ID NO: 33 Semi-nested PCR can also be performed by a combination with a nucleotide or a combination of the oligonucleotide described in SEQ ID NO: 14 and the oligonucleotide described in SEQ ID NO: 32. Moreover, the oligonucleotide which has the base sequence in any one of sequence number: 13, 14, 32 and 33 can also be used as a probe of various hybridizations, such as a microarray and electrophoresis.

(Oligonucleotide set or oligonucleotide for coronavirus detection)
Examples of the target of the coronavirus surface glycoprotein gene include primers and probes each having the base sequence set forth in SEQ ID NOs: 19, 20, 40, and 41. When these oligonucleotides are used as primers, PCR or RT-PCR is performed using the oligonucleotide set forth in SEQ ID NO: 19 as a sense primer and the oligonucleotide set forth in SEQ ID NO: 20 as an antisense primer. In this surface glycoprotein gene, 357 bases corresponding to the region sandwiched between both primers can be amplified. In addition, after performing PCR or RT-PCR using the oligonucleotide shown in SEQ ID NO: 19 and the oligonucleotide shown in SEQ ID NO: 20, the PCR product was subjected to the oligonucleotide shown in SEQ ID NO: 40. Nested PCR can also be performed using as a sense primer and the oligonucleotide set forth in SEQ ID NO: 41 as an antisense primer. Thereby, 263 bases corresponding to the region sandwiched between both primers in the coronavirus surface glycoprotein gene can be amplified. Alternatively, for the PCR product obtained by combining the oligonucleotide shown in SEQ ID NO: 19 and the oligonucleotide shown in SEQ ID NO: 20, the oligonucleotide shown in SEQ ID NO: 19 and the oligonucleotide shown in SEQ ID NO: 41 are used. Semi-nested PCR can also be performed by a combination with a nucleotide or a combination of the oligonucleotide described in SEQ ID NO: 20 and the oligonucleotide described in SEQ ID NO: 40. In addition, the oligonucleotide having the base sequence described in any one of SEQ ID NOs: 19, 20, 40 and 41 can also be used as a probe for various hybridizations such as microarrays and electrophoresis.

(Oligonucleotide set or oligonucleotide for Aichi virus detection)
Examples of the target for the VP1 gene of Aichi virus include primers and probes each having the base sequence set forth in SEQ ID NOs: 21, 22, 42 and 43. When these oligonucleotides are used as primers, by performing PCR or RT-PCR using the oligonucleotide shown in SEQ ID NO: 21 as a sense primer and the oligonucleotide shown in SEQ ID NO: 22 as an antisense primer, 395 bases corresponding to the region sandwiched between both primers in the VP1 gene can be amplified. Moreover, after performing PCR or RT-PCR using the oligonucleotide described in SEQ ID NO: 21 and the oligonucleotide described in SEQ ID NO: 22, the oligonucleotide described in SEQ ID NO: 42 with respect to the PCR product Nested PCR can also be performed using as a sense primer and the oligonucleotide set forth in SEQ ID NO: 43 as an antisense primer. As a result, 206 bases corresponding to the region sandwiched between both primers in the VP1 gene of Aichi virus can be amplified. Alternatively, for the PCR product obtained by combining the oligonucleotide described in SEQ ID NO: 21 and the oligonucleotide described in SEQ ID NO: 22, the oligonucleotide described in SEQ ID NO: 21 and the oligo described in SEQ ID NO: 43 Semi-nested PCR can also be performed by a combination with a nucleotide or a combination of the oligonucleotide described in SEQ ID NO: 22 and the oligonucleotide described in SEQ ID NO: 42. In addition, the oligonucleotide having the base sequence described in any one of SEQ ID NOs: 21, 22, 42 and 43 can be used as a probe for various hybridizations such as microarrays and electrophoresis.

  An oligonucleotide that hybridizes under stringent conditions with an oligonucleotide consisting of the base sequence described in any one of SEQ ID NOs: 1 to 43 or a base sequence complementary to a part thereof can also be used. At this time, the homology with the oligonucleotide described in any of SEQ ID NOs: 1 to 43 is preferably 80% or more, more preferably 90% or more, and still more preferably 98% in the isolated oligonucleotide. That's it.

(Detection method of diarrhea-causing virus)
In the detection method of the present invention, a sample collected from a human or non-human animal can be used as a specimen. The specimen is not particularly limited, and examples include pharyngeal wiping solution, blood, and feces, and it is preferable to use feces as the specimen.

  Moreover, the detection method of the present invention can detect diarrhea-causing virus by performing PCR or the like using the nucleotide set or oligonucleotide of the present invention. In detecting diarrhea-causing virus, after preparing a sample sample that may contain nucleic acid derived from diarrhea-causing virus, PCR is performed using the nucleotide set or oligonucleotide of the present invention as a primer, and amplification is performed by PCR. Check the marked area. In this PCR, it is preferable to perform nested PCR using the PCR product as a template after performing PCR using a specimen sample. Since the detection method of the present invention performs PCR or nested PCR using the nucleotide set or oligonucleotide of the present invention as a primer, even if plural types of primer pairs for detecting each virus are included, Each primer pair is capable of amplifying a specific region of the genome of the virus that causes diarrhea that is specifically targeted. As a result, multiple types of viruses can be detected quickly and exhaustively.

  Moreover, when confirming the region amplified by PCR, it may be confirmed by electrophoresis or by hybridization. In the electrophoresis method, it is possible to confirm whether the target gene is amplified or not by the chain length of the PCR product stained with ethidium bromide after electrophoresis. In the hybridization method, it can be confirmed whether the target region is amplified by a microarray using the oligonucleotide set or oligonucleotide of the present invention as a probe.

  In addition, specific methods for DNA or RNA extraction in the extraction step, specific methods for PCR, RT-PCR, nested PCR, etc. in the amplification step, and specific methods such as electrophoresis and microarray in the detection step are conventionally known. It can be performed according to known procedures or manufacturer's protocols.

(Use for diagnosis, etc.)
The 10 viruses according to the present invention are causative viruses of diarrhea. Therefore, after preparing a specimen sample that may contain a nucleic acid derived from a diarrhea-causing virus using a specimen such as stool from such a diarrhea patient as a test sample, PCR is performed using the nucleotide set or oligonucleotide of the present invention as a primer. By confirming the region amplified by PCR, it is possible to identify whether or not the patient is infected with any of the 10 types of diarrhea-causing viruses. This makes it possible to diagnose diarrhea. That is, a diagnostic method for diarrhea is also provided.

  Hereinafter, although the Example which actualized this invention is described, the following Examples do not limit this invention.

(Example 1: Extraction of DNA or RNA and reverse transcription of RNA)
DNA or RNA was extracted from adenovirus, enterovirus, herpes simplex virus type I, herpes simplex virus type II, group A rotavirus, group C rotavirus, astrovirus, hepatitis A virus, coronavirus and aichi virus. DNA or RNA extraction and RNA reverse transcription were performed as follows. Viruses purchased from ATCC (American Type Culture Collection) or those distributed from research institutions were used.

  The DNA of the DNA virus is obtained by centrifuging the virus culture (13,000 rpm, 20 minutes), removing the supernatant, and lysing the precipitate with Lysis buffer (0.1 M Tris-HCl, pH 7.4, 0.01 M EDTA, 0.05 M NaCl, The suspension was suspended in 0.6% SDS, 0.2 μg Proteinase K), reacted at 55 ° C. for 1 hour, treated with phenol / chloroform, and ethanol precipitated. The extracted DNA was dissolved in sterile distilled water.

  For RNA virus RNA, centrifuge the virus culture solution (13,000 rpm, 20 minutes), remove the supernatant, suspend the precipitate in sterile distilled water, and use the virus RNA extraction kit SepaGene RV-R (Sanko Junyaku) did.

  RNA reverse transcription (RT) reaction was carried out using 17 μL master mix (4 μL 5 × Buffer, 4 μL 2.5 mM dNTPs, 1 μL 50 μM random primer (TaKaRa), 1 μL 0.5 μg / μL oligo dt primer (Invitrogen)), 1.0 μL of RNA solution. μL 40u / μL RNase Inhibitor (TaKaRa), 1μL 100mM DTT, 1μL 200u / μL Super Script II RT (Invitrogen), 4.0μL sterilized distilled water) was added at 42 ° C for 1 hour, then reversed at 99 ° C for 5 minutes The enzyme was deactivated.

(Example 2: Gene amplification by multiplex PCR using primers for diarrhea-causing virus and examination thereof)
Adenovirus hexon gene, enterovirus 5 'untranslated region, herpes simplex virus type I DNA polymerase gene, herpes simplex virus type II glycoprotein gene, VP4 gene of group A rotavirus, VP4 gene of group C rotavirus, Primers were set for the astrovirus protease gene, the 5 'untranslated region of hepatitis A virus, the coronavirus surface glycoprotein gene, and the Aichi virus VP1 gene (Table 1). Primer synthesis was outsourced to Texas Genomics Japan.

  PCR was performed as follows. That is, the PCR reaction solution consists of 1 μL of extracted DNA or prepared cDNA solution and a master pool (2.5 μl BlendTaq plus Buffer, 2.5 μl 2 mM dNTP, 0.2 μl BlendTaq Plus (2.5 U / μl, manufactured by Toyobo), 1.0 μl outer primer or inner primer (25 μM), 17.8 μl DW) 24 μL was added to the PCR tube and mixed to a final volume of 25 μL. As a negative control, sterile distilled water was used in place of DNA. PCR (touchdown PCR) was performed with a gene amplifier (TaKaRa) under the following conditions. After 94 ° C 3 minutes, 94 ° C 30 seconds, 65 ° C 30 seconds, 72 ° C 30 seconds after 2 cycles, 94 ° C 30 seconds, 60 ° C 30 seconds, 72 ° C 30 seconds after 2 cycles, 94 ° C 30 seconds, 55 ° C 30 seconds, 72 ° C 30 seconds after 2 cycles, 94 ° C 30 seconds, 50 ° C 30 seconds, 72 ° C 30 seconds 30 cycles, 72 ° C 7 minutes.

  Table 1 Multiplex PCR was performed using primers for diarrhea-causing viruses. In the multiplex PCR, first, PCR was performed using a primer (outer primer mix) mixed with 22 types of outer primers (SEQ ID NOs: 1 to 22) shown in Table 1 as primers. Nested PCR was carried out using a mixture of the 24 types of inner primers (SEQ ID NOs: 4, 10, 12, 23 to 43) described in 1 (inner primer mix) as primers. The target viruses were 10 types and 17 types shown in Table 2, and after extracting DNA or RNA for each virus, cDNA was prepared for RNA, and multiplex PCR was performed in a separate PCR tube for each virus. .

  The target gene was confirmed by gel electrophoresis of the PCR product after completion of the reaction. The results of PCR using the outer primer mix are shown in FIG. 1, and the results of nested PCR using the inner primer mix are shown in FIG. As can be seen from FIG. 1 and FIG. 2, the virus genes of all 10 types and 17 strains were efficiently amplified using any primer of the outer primer mix and the inner primer mix. That is, by performing PCR using a primer mix in which all the outer primers and inner primers listed in Table 1 are mixed, a specific region of the genome of the diarrhea-causing virus that is specifically targeted is amplified. It can be said that each virus can be detected quickly and specifically. In addition, when detecting a virus, it can be expected to shorten the detection time and labor. Moreover, it was shown that detection sensitivity increases by performing nested PCR.

(Example 3: Gene amplification by PCR using adenovirus primers and examination thereof)
PCR was performed using adenovirus primers (SEQ ID NOs: 1, 2, 23, 24) among the diarrhea-causing virus primers in Table 1. For PCR, first, PCR was performed using outer primers (SEQ ID NOs: 1 and 2) as primers, and then nested PCR was performed using inner primers (SEQ ID NOs: 23 and 24) as primers. The target viruses were 23 strains shown in Table 3, DNA or RNA was extracted for each virus, cDNA was prepared for RNA, and PCR was performed in separate PCR tubes.

  The target gene was confirmed by gel electrophoresis of the PCR product after completion of the reaction. The results of PCR using the outer primer are shown in FIG. 3 (a), and the results of nested PCR using the inner primer are shown in FIG. 3 (b). As can be seen from FIG. 3, the viral genes of all 23 strains were efficiently amplified with either the outer primer or the inner primer. That is, by performing PCR using the adenovirus primers listed in Table 1, a specific region of the adenovirus genome can be specifically amplified, and adenovirus can be detected quickly and specifically. I can say that.

(Example 4: Gene amplification by PCR using primers for enterovirus and examination thereof)
PCR was performed using the enterovirus primer (SEQ ID NOs: 3, 4, 25) among the diarrhea-causing virus primers in Table 1. First, PCR was performed using the outer primer (SEQ ID NOs: 3 and 4) as a primer, and then semi-nested PCR was performed using the inner primer (SEQ ID NOs: 4 and 25) as a primer. The target viruses were 69 strains shown in Table 4, RNA was extracted from each virus, cDNA was prepared, and PCR was performed in separate PCR tubes.

  The target gene was confirmed by gel electrophoresis of the PCR product after completion of the reaction. FIG. 4 shows the results of PCR using the outer primer, and FIG. 5 shows the results of semi-nested PCR using the inner primer. As can be seen from FIGS. 4 and 5, all 69 strains of viral genes were efficiently amplified with either the outer primer or the inner primer. That is, it can be said that by performing PCR using the enterovirus primers shown in Table 1, a specific region of the enterovirus genome can be specifically amplified, and enterovirus can be detected quickly and specifically. .

(Example 5: Gene amplification by PCR using herpes simplex virus type I primer and examination thereof)
PCR was performed using the herpes simplex virus type I primer (SEQ ID NO: 5, 6, 26, 27) among the diarrhea-causing virus primers in Table 1. First, PCR was performed using outer primer (SEQ ID NO: 5 and 6) as a primer, and then nested PCR was performed using inner primer (SEQ ID NO: 26 and 27) as a primer. The target viruses were two strains shown in Table 5, and after extracting DNA or RNA for each virus, cDNA was prepared for RNA and PCR was performed in separate PCR tubes.

  The target gene was confirmed by gel electrophoresis of the PCR product after completion of the reaction. The result of PCR using the outer primer and the result of nested PCR using the inner primer are shown in FIG. As can be seen from FIG. 6 (a), all the viral genes were efficiently amplified using either the outer primer or the inner primer. That is, by performing PCR using the herpes simplex virus type I primers shown in Table 1, a specific region of the herpes simplex virus type I genome can be specifically amplified, and the herpes simplex virus type I can be rapidly It can be said that it can be detected specifically and specifically. These viral genes were not amplified by the herpes simplex virus type II primer (see lane 1 and lane 2 in FIG. 6B).

(Example 6: Gene amplification by PCR using herpes simplex virus type II primer and examination thereof)
PCR was performed using herpes simplex virus type II primers (SEQ ID NOs: 7, 8, 28, 29) among the primers for diarrhea-causing viruses in Table 1. First, PCR was performed using outer primer (SEQ ID NO: 7 and 8) as a primer, and then nested PCR was performed using inner primer (SEQ ID NO: 28 and 29) as a primer. The target viruses were the two strains shown in Table 6, and after extracting DNA or RNA for each virus, cDNA was prepared for RNA and PCR was performed in separate PCR tubes.

  The target gene was confirmed by gel electrophoresis of the PCR product after completion of the reaction. The result of PCR using the outer primer and the result of nested PCR using the inner primer are shown in FIG. As can be seen from FIG. 6 (b), all the viral genes were efficiently amplified using either the outer primer or the inner primer. That is, by performing PCR using the herpes simplex virus type II primers shown in Table 1, a specific region of the herpes simplex virus type II genome can be specifically amplified. It can be said that it can be detected specifically and specifically. These viral genes were not amplified by the herpes virus type I primer (see lane 3 and lane 4 in FIG. 6 (a)).

(Example 7: Gene amplification by PCR using primers for group A rotavirus and examination thereof)
PCR was performed using primers for group A rotavirus (SEQ ID NOs: 9, 10, 11, 12, 30, 31) among the primers for diarrhea-causing viruses in Table 1. First, PCR is performed using a mixed primer obtained by mixing four types of outer primers (SEQ ID NOs: 9, 10, 11, 12), and then four types of inner primers (SEQ ID NOs: 10, 12, 30). , 31) was used to perform semi-nested PCR. The target viruses were 7 strains shown in Table 7, RNA was extracted from each virus, cDNA was prepared, and PCR was performed in separate PCR tubes.

  The target gene was confirmed by gel electrophoresis of the PCR product after completion of the reaction. The results of PCR using the outer primer and the results of semi-nested PCR using the inner primer are shown in FIG. In FIG. 7, (a) shows the use of the outer primer, and (b) shows the use of the inner primer. As can be seen from FIG. 7, the virus genes of all seven strains were efficiently amplified with either the outer primer or the inner primer. That is, by performing PCR using the primers for group A rotavirus shown in Table 1, a specific region of the genome of group A rotavirus can be specifically amplified, and the group A rotavirus can be rapidly and specifically identified. It can be said that it can be detected automatically.

(Example 8: Gene amplification by PCR using primers for group C rotavirus and examination thereof)
PCR was performed using primers for group C rotavirus (SEQ ID NOs: 13, 14, 32, 33) among the primers for diarrhea-causing viruses in Table 1. First, PCR was performed using outer primer (SEQ ID NO: 13, 14) as a primer, and then nested PCR was performed using inner primer (SEQ ID NO: 32, 33) as a primer. After extracting RNA from one target virus strain, cDNA was prepared and PCR was performed.

  The target gene was confirmed by gel electrophoresis of the PCR product after completion of the reaction. The results of PCR using the outer primer and the results of nested PCR using the inner primer are shown in FIG. As can be seen from FIG. 7, the viral gene was efficiently amplified when both the outer primer and the inner primer were used. That is, by performing PCR using the primers for Group C rotavirus shown in Table 1, a specific region of the C-type rotavirus genome can be specifically amplified, and the group C rotavirus can be rapidly and specifically identified. It can be said that it can be detected automatically.

(Example 9: Gene amplification by PCR using primers for astrovirus and examination thereof)
PCR was performed using astrovirus primers (SEQ ID NOs: 15, 16, 34, 35, 36, and 37) among diarrhea-causing virus primers in Table 1. First, PCR is performed using outer primer (SEQ ID NO: 15 and 16) as a primer, and then nested PCR is performed using inner primer (a combination of SEQ ID NO: 34, 35 or a combination of SEQ ID NOs: 36, 37) as a primer. went. The target viruses were 8 strains shown in Table 8, RNA was extracted from each virus, cDNA was prepared, and PCR was performed in separate PCR tubes.

  The target gene was confirmed by gel electrophoresis of the PCR product after completion of the reaction. The results of PCR using the outer primer are shown in FIG. 8 (a), the results of nested PCR using SEQ ID NO: 34, 35 as the inner primer are shown in FIG. 8 (b), and SEQ ID NO: 36, The result of nested PCR using 37 is shown in FIG. As can be seen from FIGS. 8 (a) to 8 (c), all the viral genes were efficiently amplified in the case of using either the outer primer or the inner primer. That is, by performing PCR using the astrovirus primers shown in Table 1, a specific region of the astrovirus genome can be specifically amplified, and astrovirus can be detected quickly and specifically. I can say that.

(Example 10: Gene amplification by PCR using primers for hepatitis A virus and examination thereof)
PCR was performed using primers for hepatitis A virus (SEQ ID NOs: 17, 18, 38, 39) among primers for diarrhea-causing viruses in Table 1. First, PCR was performed using outer primer (SEQ ID NO: 17, 18) as a primer, and then nested PCR was performed using inner primer (SEQ ID NO: 38, 39) as a primer. The target viruses were the two strains shown in Table 9, RNA was extracted from each virus, cDNA was prepared, and PCR was performed in separate PCR tubes.

  The target gene was confirmed by gel electrophoresis of the PCR product after completion of the reaction. The result of PCR using the outer primer and the result of nested PCR using the inner primer are shown in FIG. As can be seen from FIG. 9 (a), all the viral genes were efficiently amplified using either the outer primer or the inner primer. That is, by performing PCR using the hepatitis A virus primers listed in Table 1, a specific region of the hepatitis A virus genome can be specifically amplified, and hepatitis A virus can be rapidly and specifically identified. It can be said that it can be detected automatically.

(Example 11: Gene amplification by PCR using primers for coronavirus and examination thereof)
PCR was performed using coronavirus primers (SEQ ID NOs: 19, 20, 40, 41) among the diarrhea-causing virus primers in Table 1. First, PCR was performed using outer primer (SEQ ID NO: 19, 20) as a primer, and then nested PCR was performed using inner primer (SEQ ID NO: 40, 41) as a primer. RNA was extracted from one target virus strain (coronavirus OC43), cDNA was prepared, and PCR was performed.

  The target gene was confirmed by gel electrophoresis of the PCR product after completion of the reaction. The result of PCR using the outer primer and the result of nested PCR using the inner primer are shown in FIG. 9B. As can be seen from FIG. 9 (b), the viral gene was efficiently amplified when both the outer primer and the inner primer were used. That is, by performing PCR using the coronavirus primers listed in Table 1, a specific region of the coronavirus genome can be specifically amplified, and coronavirus can be detected quickly and specifically. I can say that.

(Example 12: Gene amplification by PCR using primers for Aichi virus and examination thereof)
PCR was performed using primers for ichi virus (SEQ ID NOs: 21, 22, 42, 43) among the primers for diarrhea-causing viruses in Table 1. For PCR, first, PCR was performed using outer primers (SEQ ID NOs: 21 and 22) as primers, and then nested PCR was performed using inner primers (SEQ ID NOs: 42 and 43) as primers. After extracting RNA from one target virus strain, cDNA was prepared and PCR was performed.

  The target gene was confirmed by gel electrophoresis of the PCR product after completion of the reaction. The result of PCR using the outer primer and the result of nested PCR using the inner primer are shown in FIG. 9B. As can be seen from FIG. 9 (b), the viral gene was efficiently amplified when both the outer primer and the inner primer were used. That is, by performing PCR using the primers for Aichi virus shown in Table 1, a specific region of the Aichi virus genome can be specifically amplified, and Aichi virus can be detected quickly and specifically. I can say that.

  SEQ ID NOs: 1-43: Synthetic DNA

The figure which shows the result of carrying out the gel electrophoresis of the PCR product of multiplex PCR using outer primer mix. The figure which shows the result of carrying out the gel electrophoresis of the PCR product of multiplex PCR using inner primer mix. The figure which shows the result of carrying out gel electrophoresis of the PCR product at the time of using the primer for adenovirus. The figure which shows the result of carrying out the gel electrophoresis of the PCR product at the time of using the primer for enteroviruses (outer primer). The figure which shows the result of having carried out the gel electrophoresis of the PCR product at the time of using the primer for enteroviruses (inner primer). The figure which shows the result of having carried out the gel electrophoresis of the PCR product at the time of using the primer for herpesviruses. The figure which shows the result of carrying out gel electrophoresis of the PCR product at the time of using the primer for A group rotavirus or the primer for C group rotavirus. The figure which shows the result of carrying out gel electrophoresis of the PCR product at the time of using the primer for astrovirus. The figure which shows the result of carrying out gel electrophoresis of the PCR product at the time of using the primer for hepatitis A virus, the primer for coronavirus, or the primer for Aichi virus.

Claims (19)

An oligonucleotide set for detecting a diarrhea-causing virus,
Adenovirus hexon gene, enterovirus 5 'untranslated region, herpes simplex virus type I DNA polymerase gene, herpes simplex virus type II glycoprotein gene, group A rotavirus VP4 gene, group C rotavirus VP4 gene, A polynucleotide encoding at least two regions selected from an astrovirus protease gene, a 5 ′ untranslated region of hepatitis A virus, a surface glycoprotein gene of coronavirus, and a VP1 gene of Aichi virus, or a part thereof An oligonucleotide set comprising oligonucleotides that hybridize.   The oligonucleotide set according to claim 1, wherein the oligonucleotide set comprises five or more kinds of the oligonucleotides.   The oligonucleotide set according to claim 1, wherein the oligonucleotide set comprises eight or more kinds of the oligonucleotides.   The oligonucleotide set according to any one of claims 1 to 3, wherein the oligonucleotide set detects two or more diarrhea-causing viruses simultaneously. The oligonucleotide set according to any one of claims 1 to 3, wherein the oligonucleotide set includes two or more kinds of oligonucleotides selected from the group consisting of the following (a) to (j).
(A) a base sequence set forth in SEQ ID NO: 1, a base sequence set forth in SEQ ID NO: 2, a base sequence set forth in SEQ ID NO: 23, a base sequence set forth in SEQ ID NO: 24, and those in an adenovirus hexon gene One or more oligonucleotides targeting a region sandwiched by any two base sequences of the complementary sequences of (b) The base sequence and sequence set forth in SEQ ID NO: 3 in the 5 ′ untranslated region of enterovirus One or more oligonucleotides targeting a region sandwiched by any two base sequences of the base sequence described in No. 4 and the base sequence described in SEQ ID NO: 25 and their complementary sequences (c ) The nucleotide sequence set forth in SEQ ID NO: 5 in the herpes simplex virus type I DNA polymerase gene, the nucleotide sequence set forth in SEQ ID NO: 6, One or more oligonucleotides that target a region sandwiched by any two base sequences of the base sequence set forth in column number: 26, the base sequence set forth in SEQ ID NO: 27, and their complementary sequences ( d) The base sequence set forth in SEQ ID NO: 7, the base sequence set forth in SEQ ID NO: 8, the base sequence set forth in SEQ ID NO: 28, the base set forth in SEQ ID NO: 29 in the herpes simplex virus type II glycoprotein gene One or more oligonucleotides targeting a region sandwiched between any two of the sequences and their complementary sequences (e) the base set forth in SEQ ID NO: 9 in the VP4 gene of group A rotavirus Sequence, base sequence set forth in SEQ ID NO: 10, base sequence set forth in SEQ ID NO: 11, base sequence set forth in SEQ ID NO: 12, SEQ ID NO: 30 1 type or 2 types or more of oligonucleotides which target the area | region pinched | interposed by any two base sequences among the base sequence of description, sequence number: 31, and those complementary sequences (f) C group rota In the viral VP4 gene, the nucleotide sequence of SEQ ID NO: 13, the nucleotide sequence of SEQ ID NO: 14, the nucleotide sequence of SEQ ID NO: 32, the nucleotide sequence of SEQ ID NO: 33, and their complementary sequences One or more oligonucleotides targeting a region sandwiched by any two of these nucleotide sequences (g) The nucleotide sequence of SEQ ID NO: 15 in the protease gene of astrovirus, the sequence of SEQ ID NO: 16 Base sequence, base sequence set forth in SEQ ID NO: 34, base sequence set forth in SEQ ID NO: 35, base sequence set forth in SEQ ID NO: 36, One or more oligonucleotides targeting a region sandwiched by any two nucleotide sequences of the nucleotide sequence set forth in SEQ ID NO: 37 and their complementary sequences (h) 5 ′ non-hepatitis A virus Any of the base sequence set forth in SEQ ID NO: 17 in the translation region, the base sequence set forth in SEQ ID NO: 18, the base sequence set forth in SEQ ID NO: 38, the base sequence set forth in SEQ ID NO: 39, and their complementary sequences One or two or more oligonucleotides targeting a region sandwiched between two base sequences (i) The base sequence described in SEQ ID NO: 19 in the coronavirus surface glycoprotein gene, described in SEQ ID NO: 20 Any of the base sequence, the base sequence set forth in SEQ ID NO: 40, the base sequence set forth in SEQ ID NO: 41, and their complementary sequences One or more oligonucleotides targeting a region sandwiched by two base sequences (j) The base sequence set forth in SEQ ID NO: 21 in the VP1 gene of Aichi virus, the base sequence set forth in SEQ ID NO: 22, 1 type or 2 or more types of oligonucleotide which targets the area | region pinched | interposed by any two base sequences among the base sequence of number: 42, the base sequence of sequence number: 43, and those complementary sequences The one or more oligonucleotides of (a) are the nucleotide sequence set forth in SEQ ID NO: 1, the nucleotide sequence set forth in SEQ ID NO: 2, the nucleotide sequence set forth in SEQ ID NO: 23, and the SEQ ID NO: 24. Including oligonucleotides each having one or more base sequences selected from the base sequences described in
1 type or 2 types or more of said oligonucleotide of (b) is selected from the base sequence of sequence number: 3, the base sequence of sequence number: 4, and the base sequence of sequence number: 25 Including oligonucleotides each having a species or two or more nucleotide sequences,
The one or more oligonucleotides of (c) are the base sequence shown in SEQ ID NO: 5, the base sequence shown in SEQ ID NO: 6, the base sequence shown in SEQ ID NO: 26, and the SEQ ID NO: 27. Including oligonucleotides each having one or more base sequences selected from the base sequences described in
One or two or more oligonucleotides of (d) are the base sequence shown in SEQ ID NO: 7, the base sequence shown in SEQ ID NO: 8, the base sequence shown in SEQ ID NO: 28, and the sequence number 29. Including oligonucleotides each having one or more base sequences selected from the base sequences described in
The one or more oligonucleotides of (e) above are the base sequence shown in SEQ ID NO: 9, the base sequence shown in SEQ ID NO: 10, the base sequence shown in SEQ ID NO: 11, and the sequence number: 12 Comprising oligonucleotides each having one or more base sequences selected from the base sequence described in SEQ ID NO: 30, the base sequence set forth in SEQ ID NO: 30 and the base sequence set forth in SEQ ID NO: 31;
One or more oligonucleotides of the above (f) are the base sequence shown in SEQ ID NO: 13, the base sequence shown in SEQ ID NO: 14, the base sequence shown in SEQ ID NO: 32, and the SEQ ID NO: 33. Including oligonucleotides each having one or more base sequences selected from the base sequences described in
The one or more oligonucleotides of (g) are the nucleotide sequence set forth in SEQ ID NO: 15, the nucleotide sequence set forth in SEQ ID NO: 16 and the nucleotide sequence set forth in SEQ ID NO: 34, SEQ ID NO: 35. Comprising oligonucleotides each having one or more base sequences selected from the base sequence set forth in SEQ ID NO: 36, the base sequence set forth in SEQ ID NO: 36, and the base sequence set forth in SEQ ID NO: 37;
The one or more oligonucleotides of (h) are the nucleotide sequence set forth in SEQ ID NO: 17, the nucleotide sequence set forth in SEQ ID NO: 18, the nucleotide sequence set forth in SEQ ID NO: 38, and the sequence number: 39. Including oligonucleotides each having one or more base sequences selected from the base sequences described in
The one or more oligonucleotides of (i) above are the base sequence shown in SEQ ID NO: 19, the base sequence shown in SEQ ID NO: 20, the base sequence shown in SEQ ID NO: 40, the sequence number: 41. Including oligonucleotides each having one or more base sequences selected from the base sequences described in
The one or more oligonucleotides of (j) above are the base sequence set forth in SEQ ID NO: 21, the base sequence set forth in SEQ ID NO: 22, the base sequence set forth in SEQ ID NO: 42, and the SEQ ID NO: 43. Including oligonucleotides each having one or more base sequences selected from the base sequences described in 1.
The oligonucleotide set according to claim 5.   The oligonucleotide set according to any one of claims 1 to 6, wherein the oligonucleotide is a primer.   The oligonucleotide set according to any one of claims 1 to 6, wherein the oligonucleotide is a probe. An oligonucleotide for detecting a diarrhea-causing virus selected from the group consisting of the following (A) to (J).
(A) One or two selected from the base sequence set forth in SEQ ID NO: 1, the base sequence set forth in SEQ ID NO: 2, the base sequence set forth in SEQ ID NO: 23, and the base sequence set forth in SEQ ID NO: 24 Oligonucleotide for detecting adenovirus each having a base sequence of at least species (B) From the base sequence set forth in SEQ ID NO: 3, the base sequence set forth in SEQ ID NO: 4 and the base sequence set forth in SEQ ID NO: 25 Oligonucleotide for detecting an enterovirus having one or more selected base sequences, respectively (C) The base sequence described in SEQ ID NO: 5, the base sequence described in SEQ ID NO: 6, and the sequence number: 26 For detecting herpes simplex virus type I each having one or more base sequences selected from the base sequence described in SEQ ID NO: 27 Gotonucleotide (D) One selected from the base sequence set forth in SEQ ID NO: 7, the base sequence set forth in SEQ ID NO: 8, the base sequence set forth in SEQ ID NO: 28, and the base sequence set forth in SEQ ID NO: 29 Or an oligonucleotide for detecting herpes simplex virus type II each having two or more types of base sequences (E) the base sequence set forth in SEQ ID NO: 9, the base sequence set forth in SEQ ID NO: 10 and the SEQ ID NO: 11 Each having one or more base sequences selected from the base sequence described, the base sequence set forth in SEQ ID NO: 12, the base sequence set forth in SEQ ID NO: 30, and the base sequence set forth in SEQ ID NO: 31 Oligonucleotide for Detecting Group A Rotavirus (F) The base sequence described in SEQ ID NO: 13, the base sequence described in SEQ ID NO: 14, the base sequence described in SEQ ID NO: 32, and Oligonucleotide for detecting group C rotavirus each having one or more base sequences selected from the base sequence set forth in column number 33: (G) the base sequence and sequence set forth in SEQ ID NO: 15 From the base sequence set forth in No. 16; the base sequence set forth in SEQ ID NO: 34; the base sequence set forth in SEQ ID NO: 35; the base sequence set forth in SEQ ID NO: 36; and the base sequence group set forth in SEQ ID NO: 37 Oligonucleotide for detecting astrovirus each having one or more selected base sequences (H) Base sequence described in SEQ ID NO: 17, base sequence described in SEQ ID NO: 18, SEQ ID NO: 38 for detecting hepatitis A virus each having one or more base sequences selected from the base sequence set forth in SEQ ID NO: 38 and the base sequence set forth in SEQ ID NO: 39. Ligonucleotide (I) One selected from the base sequence set forth in SEQ ID NO: 19, the base sequence set forth in SEQ ID NO: 20, the base sequence set forth in SEQ ID NO: 40, and the base sequence set forth in SEQ ID NO: 41 Or an oligonucleotide for detecting a coronavirus each having two or more base sequences (J) the base sequence set forth in SEQ ID NO: 21, the base sequence set forth in SEQ ID NO: 22 and the base set forth in SEQ ID NO: 42 Oligonucleotide for detecting Aichi virus each having one or more base sequences selected from the sequence and the base sequence set forth in SEQ ID NO: 43   The oligonucleotide for detecting diarrhea-causing virus according to claim 9, wherein the oligonucleotide is a primer.   The oligonucleotide for detecting diarrhea-causing virus according to claim 9, wherein the oligonucleotide is a probe.   A solid phase carrier in which the oligonucleotide contained in the oligonucleotide set according to any one of claims 1 to 8 or the oligonucleotide according to any one of claims 9 to 11 is immobilized on a solid support.   A detection kit for detecting a diarrhea-causing virus comprising the oligonucleotide set according to any one of claims 1 to 8 or the oligonucleotide according to any one of claims 9 to 11. Preparing a specimen sample that may contain nucleic acid derived from a diarrhea-causing virus;
PCR step of performing PCR using the nucleic acid as a template and the oligonucleotide set according to claim 7 or the oligonucleotide according to claim 10 as a primer;
A detection step for detecting a PCR product obtained by the PCR step;
A method for detecting a virus caused by diarrhea, comprising:   The method for detecting a diarrhea-causing virus according to claim 14, wherein the detection step uses the oligonucleotide set according to claim 8 or the oligonucleotide according to claim 11 as a probe when detecting the PCR product.   The method for detecting a diarrhea-causing virus according to claim 14 or 15, wherein the PCR includes nested PCR. A method for diagnosing diarrhea,
Preparing a sample sample that may contain nucleic acid derived from a diarrhea-causing virus from a test sample collected from an animal; and
PCR step of performing PCR using the nucleic acid as a template and the oligonucleotide set according to claim 6 or the oligonucleotide according to claim 11 as a primer;
A detection step for detecting a PCR product obtained by the PCR step;
A diagnostic step of diagnosing either the risk of developing the diarrhea of the animal based on the detection result of the detection step, whether the animal is suffering from diarrhea and the degree of progression of the diarrhea;
A method for diagnosing diarrhea, comprising:   The method for diagnosing diarrhea according to claim 17, wherein the detection step uses the oligonucleotide set according to claim 8 or the oligonucleotide according to claim 11 as a probe when detecting the PCR product.   The method for diagnosing a diarrhea-causing virus according to claim 17 or 18, wherein the PCR includes a nested PCR.