TW200804604A - Detection method for koi herpes virus (KHV) - Google Patents

Abstract

It is intended to provide: a primer set for detecting Koi herpes virus whereby Koi herpes virus infecting carp can be more accurately and quickly detected and identified; a probe for detecting Koi herpes virus; and a method of detecting Koi herpes virus by using the above-described primer set and probe. By using a primer set for detecting Koi herpes virus (KHV) characterized by having a first DNA primer which contains an oligonucleotide part having at least 10 bases in the base sequence represented by SEQ ID NO:1 and a second DNA primer which contains an oligonucleotide part having at least 10 bases in the base sequence represented by SEQ ID NO:2, Koi herpes virus can be more accurately and quickly detected and identified.

Description

200804604 IX. OBJECTS OF THE INVENTION: TECHNICAL FIELD The present invention relates to a primer set for detecting a cancer virus of a koi herpes virus (khv) which is infected by a koi, and a test for detecting a cancer virus A needle, and a method for detecting a cancer virus using the primer set or probe. [Prior Art] Jin, acne virus disease, which is a disease caused by ..., "li" or koi due to infection of jinfu vesicle virus (khv). If the disease occurs, it is sick of black carp or koi. The action becomes sluggish 'and does not eat bait food, but significant external symptoms), it can be seen that the fading or erosion (festering) of the ankle, etc., occurs from the fry to the adult fish and the mortality rate is high. The above-mentioned Koi herpes virus disease, in 1998 The disease was first reported in Israel in May. Since then, the same in Israel, in the fall and the following spring, two people, including the export of koi, about 600 tasmines, all died. The total loss exceeded $4 million. Since then, reports of morbidity have been reported in countries around the world (Israel, Britain, Germany, the Netherlands, Belgium, the United States, Indonesia, and Taiwan). Sakamoto also announced that in November 2003, the Ministry of Agriculture, Forestry and Fisheries Xiapu in the sub-city, the koi, suspected of suffering from the koi herpes virus disease, was confirmed, and then reported in the Aomori, Yamanashi, Mie, Okayama, Miyazaki and various places. The Ministry of Agriculture, Forestry and Fisheries is beginning to determine the route of infection and strive to prevent the spread of the disease. & When an infection of the medicinal cancer virus occurs in nature or in a farm, 119552.doc 200804604 疋 becomes the causative agent of the disease, preventing infection It is extremely important to spread or prevent the treatment of infectious diseases. Previously, including fish pathogens, when identifying common bacteria, viruses, molds, etc., observe the morphology, biochemical traits, or use immunological methods to biochemical traits. Observation. Recently, with the development of biochemistry and molecular genetics, it has been possible to identify and detect the metabolism of chromosomal DNA or RNA, pathogen constituents or pathogens of pathogens. However, these methods must be The problem of spending a lot of time after a lot of complicated steps, and the inability to adequately cope with the rapid spread of diseases such as viral diseases. " In view of such circumstances, the method of detecting specific genes in various pathogens and identifying pathogens recently It is becoming popular. This method uses ... to become the target pathogen A DNA probe (manufactured by a labeled compound, single-stranded DN A) is hybridized only to a nucleic acid which is a target pathogen. Further, if a method for mass-amplifying a target DNA in a very short time is developed, By using one of the two ends of the base sequence of the above-described identified gene as a primer, the test sample (the biological tissue containing the pathogen) is used to quickly and accurately detect and identify a pathogen such as a virus. There are purely virus type and type-specific detection methods. 1 There are known methods or substances: L-έ can be used to determine the degree of a-form and the specificity of the type is not reduced. DNA primers and probes, which are highly accurate and rapid, and type U recognizes infections of HSVI type or HSVII type, so that the results are useful for diagnosis and treatment of m treatment (for example, refer to the patent literature). Or a human immunodeficiency virus that is used as a cause of AIDS (5) v) H9552.doc 200804604 Infected nucleic acid, which is a nucleoside that can be used as a primer and probe in the amplification and detection of HIV-1 nucleic acid. Acid sequence (for example, refer to Patent Document 2); a method for high-sensitivity detection of yolk vegetative leaf curl virus (TYLCV) as a pathogenic virus for early diagnosis of tomato yellow leaf curl disease (for example, reference patent) Document 3.); or use the RN A containing the RN A in the wilt virus with its wilt virus or its sequence, and the DnA-part of the uracil is replaced with the RN A, which is complementary to the RN A with the stupid bean wither virus. A method of detecting a broad bean wilt virus by using a nucleic acid primer of a part of the acid of the acid (for example, refer to Patent Document 4). In addition, as for the introduction of virus detection for fish, there is proposed a method of selecting a pathogenic virus gene using a mixed primer, which uses a fish pathogenic virus such as rainbow virus, and a ribonucleotide reductase stored between virus species. In the gene, the base sequence of the amino acid sequence corresponding to the region with high preservability is designed, and then, the specific base sequence design for the fish disease diagnosis which is available in the obtained DNA is designed. A method in which a primer is used to rapidly diagnose a fish disease pathogenic rainbow virus (for example, refer to Patent Document 5). Further, a method for detecting a koi herpes virus using a PCR-amplified DNA of 292 bp (see, for example, Non-Patent Document 1) has been reported, but the manufacturer has pointed out that there are problems in detection sensitivity and accuracy. [Patent Document 1] Japanese Patent Laid-Open Publication No. JP-A No. Hei. No. Hei. No. Hei. 119552.doc 200804604

Patent Document 5: Japanese Patent Laid-Open No. Hei 7-284399, Non-Patent Document 1: Yuasa, K. et al. (2005) Fish path QlQ gy 40(1)·· 37-39 〇 [Summary of the Invention] The problem is that the koi herpes virus disease has a high mortality rate. Because the koi pimple virus disease not only causes significant damage to the koi and the natural interest in the nature of the lake, it is urgently expected to develop a more accurate and rapid detection. Methods. An object of the present invention is to provide a probe for detecting koi herpesvirus infected by koi, a probe group for detecting koi blister virus, and a probe for necropsy cancer detection, or using the primer group, which can detect and identify the koi herpes virus infected by koi more accurately and rapidly. Or the detection method of the koi herpes virus of the probe. MEANS FOR SOLVING THE PROBLEMS The inventors of the present invention have made an effort to solve the above problems, and have determined the entire base sequences of the koi herpesvirus genes of Japan, the United States, Israel, and Indonesia, and confirmed that the koi herpesviruses are at the same level of genes. The source is about 99% and the homology to the amino acid level is about 1%. It was then found that in all the well-defined sequence of the sequence, focusing on the specific repeat portion of the virus pathogen, the length of the hypothesis in searching for known eukaryotes or viruses is 15 () to 2 () () bp or so, and the sequence of the test and the composition ratio of G is not more than 65%. In Japan, the United States, Israel, and Indonesia, the specificity and (4) good sequence are selected. The two regions disclosed in Nos. 5 and 6 can be more accurately and quickly detected and identified if they use the probe 119552.doc 200804604 or the koi herpes virus detection probe using the region as the target. The Koi herpes virus finally completes the present invention. That is, the present invention relates to a (1) koi herpesvirus (KHV) detection guide, a sub-group characterized in that it has a first DNA primer and a second DNA primer, and the first DNA primer contains a sequence listing. An oligonucleotide portion of at least 10 bases of the nucleotide sequence represented by SEQ ID NO: 1, the second DNA primer comprising an oligonucleotide of at least 1 base sequence represented by SEQ ID NO: 2 in the Sequence Listing a nucleotide moiety; or (2) a primer set for detection of koi herpesvirus (KHV), characterized in that it has a first DNA primer and a second DNA primer, and the first DNA primer contains SEQ ID NO: 3 in the sequence listing. a nucleotide portion of at least 10 nucleotides of the base sequence, the second DNA primer comprising an oligonucleotide portion of at least 1 base of the base sequence shown in SEQ ID NO: 4 of the Sequence Listing; or (3) A method for detecting koi herpesvirus (KHV), characterized in that it uses the primer set for koi herpes disease (KHV) detection disclosed in the above (1) or (2) to amplify a sample to be tested. The koi herpesvirus (KHv) gene; or (4) the detection of koi herpesvirus (KHV) , characterized in that it comprises a labeled nuclear (four) portion of at least one of the base sequences of sequence number 5 of the sequence listing or its complementary sequence; or (5) brocade (four) therapeutic virus (with) Detecting a (four) needle characterized in that it has at least 1 () of the nucleotide sequence of the sequence of the sequence shown in SEQ ID NO: 6 of the sequence listing or its complement, or (6) m (d) poison ( KHv) detection:: It is: under the condition of hybridization, the above-mentioned KHV detection probe and the test sample are cultured. 0 119552.doc 200804604 Further, the present invention relates to: (7) DNA comprising a nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: or a complement thereof; or (8) DNA comprising one or more bases in the nucleotide sequence shown in Sequence 5 or 6. a base sequence deleted, replaced or added, and used as a probe to detect koi herpesvirus (KHV); or (9) DNA under stringent conditions, and containing SEQ ID NO: 5 or 6 The DNA of the complementary sequence of the indicated base sequence is hybridized, and when used as a probe, the cancer virus can be detected in the carp KHV). [Embodiment] As a primer set for detecting koi herpesvirus (KHV) of the present invention, if the first DNA primer and the second DNA primer are provided, the first DNA primer includes at least one of the sequence numbers 1 of the sequence listing. The nucleus of the base of the base sequence I part 'the second DNA primer contains an oligonucleotide portion of at least 1 碱基 of the base sequence of SEQ ID NO: 2 of the sequence listing; or has the first DNA a primer and a second DNA primer, the first DNA primer comprising an oligonucleotide of at least one base sequence of SEQ ID NO: 3 in the sequence listing, and the second DN A primer comprising the sequence of the sequence listing The oligonucleotide of at least 10 bases of the sequence of the sequence shown in No. 4 is not particularly limited, and the koi in the sample to be tested is amplified by using the primer set for detecting the koi virus. Herpes virus gene, which can detect koi herpes virus. As the first DNa primer or the second DNA primer having the nucleotide portion of at least 10 bases, it is preferred to contain an oligonucleotide of 10 to 50 bases, more preferably 15 to 15 A 30-base oligonucleotide portion, although a primer having a 20-base nucleotide portion is preferred, as long as the length of the primer is 1 in the DNA amplified by PCr H9552.doc _ 11 - 200804604 Anyone above the base can be removed. In addition, the probe for detecting koi herpesvirus (KHV) of the present invention has the nucleotide sequence shown in SEQ ID NO: 5 of the Sequence Listing (190 bp of the genomic 7689-7878 or 280527-280716 of the Koi herpes virus). a probe of at least 10 bases, preferably 20 bases or more of the labeled oligonucleotide moiety, or a base sequence of SEQ ID NO: 6 in the sequence listing; Detecting at least 10 base W groups of the koi herpesvirus genome 15996-16160 or 288834-288998 or its complementary sequence, preferably 20 bases or more of labeled oligonucleotides The needle is not particularly limited, and the probe can be detected by a usual method by culturing the probe for detecting the venom virus and the sample to be tested under the conditions of hybridization. The sample to be tested is not particularly limited as long as it is suspected to contain a sample of the koi herpes virus. For example, it can be specifically exemplified by extracting tissue or blood from the koi, epidermis, liver, kidney, etc. by the common method. DN A φ extract. The method for detecting a koi herpes virus of the present invention using the primer group for detecting cancer by the cancer of the present invention, comprising the step of amplifying the koi herpes virus DNA in the sample to be tested, and the DNA detection step of the koi herpes virus gene; Step. In the amplification step of the gene DNA, a polymerase chain reaction (PCR) method, a loop mediated Isothermal Amplification (LAMP), an Isothermal and Chimeric primer-initiated Amplification method (ICAN) can be used. Wait. 119552.doc -12- 200804604 For example, if PCR is used, only the target DNA region can be automatically amplified from trace amounts of DNA to about 1 million times (science, 239: 487-491, 1988). In the PCR, two kinds of primers for the sense strands (hereinafter referred to as the first primer) and the primers for the antisense strand (hereinafter referred to as the second primer) which are amplified in the 1) region can be used. DNA primers to amplify the Koi herpes virus gene DNA in the test sample. The respective base groups constituting the first primer or the second primer described above may be modified (for example, biotinylation or labeling with a luminescent substance) in any known state. In the DNA amplification step of PCR, the amplification cycle is repeated using the first primer and the second primer, and the DNA polymerase, preferably heat-resistant 〇>1 human polymerase. As the heat-resistant polymerase, it is particularly preferable to use a DNA polymerase which can maintain its activity up to 95 degrees, for example, a commercially available τ called polymerase. The amount of use of the first primer, the second primer, and the DNA polymerase varies depending on the type of the sample to be tested, but can be easily determined within the range in which the dna amplification step by the PCR method can be carried out. The mixture may contain a buffer (for example, Tris-HCl buffer), a stabilizer (for example, gelatin), or a salt (for example, sodium carbonate) depending on the case. The amplification conditions in the case of performing PCR using a mixture containing the primer set of the present invention, the DNA polymerase, and the test sample can be preferably exemplified by modification of DNA (about 9 Torr to 95 ° C). Next, about 1 second to about 2 minutes), the single-stranded DNA is bonded to the first primer and the second primer (about 37 to 70. (:, about 3 seconds to about 3 minutes), and using DNA polymerase ^^八合成(about 65~80C, about 30 seconds~about 5 minutes), it is better to repeat the amplification cycle of 1〇~6〇 times, especially 2〇~4〇 times. In the circulation, the preferred 119552.doc •13-200804604 is to extend the heating time of the DNA synthesis using DNA polymerase to about 5 to 10 minutes to complete the DNA synthesis. The presence of the Koi herpes virus in the sample to be tested At the end of the amplification cycle, a large amount of DNA derived from the Jinjing sore virus is synthesized, and the DNA is detected by a DNA detection step described later. As a DNA detection step, gel electrophoresis and bromide b can be used. Ingot dyeing method, using Southern Blot Hybrid method or The base sequence determination method, the radioactive labeling method, etc. of the deoxygenation method. When performing the gel electrophoresis method, for example, a latent electrophoresis using an agarose gel as a carrier or a plate-like electrophoresis using acrylamide may be used. For the case of Southern blot hybridization or in situ hybridization, radioactive probes, non-radioactive probes (eg, enzyme-labeled probes, biotinylated probes, digoxingenin) can be used. a needle, or a probe labeled with a chemiluminescent substance or a fluorescent substance. Further, in the case of using the base sequence determining method by the dideoxy method, a fluorescent nucleic acid automatic nucleic acid sequencer can be used ( Applied Biosystems, Inc. As a combination of the first primer and the second primer used in the above amplification step, a nucleotide comprising the nucleotide sequence of SEQ ID NO: 1 (acagtgtccgacttgtgcga) is preferably exemplified (hereinafter, In some cases, it is sometimes referred to as KHV-TNFR-F) and a combination of nucleotides (hereinafter referred to as KHV-TNFR-R) including the nucleotide sequence of SEQ ID NO: 2 (hereinafter referred to as KHV-TNFR-R) (hereinafter sometimes referred to as KHV-TNFR-R) KHV-TN FR primer set; or a nucleotide comprising the sequence of the sequence shown in SEQ ID NO: 3 (gacactgaacatgaacactg) (hereinafter, sometimes referred to as KHV-GT-F), and contains the sequence number 4 119552.doc -14· 200804604 A combination of oligonucleotides (hereinafter referred to as KHV-GT-R) of the base sequence (gaatccatccccatcacccg) shown (hereinafter sometimes referred to as KHV-GT primer group) may be used alone. Either one or two can be used at the same time.

The above KHV-TNFR-F is present in the sequence of the genome of the Koi herpes virus, 7689-7708 or 280527-280546, and the KHV-TNFR-R is present in the complementary strand of the sequence of the genome of the Koi herpes virus, 7859-7878 or 280697-280716; When these KHV-TNFR primer sets are used, DNA containing the 190 bp length sequence shown by SEQ ID NO: 5 can be amplified. Further, the above KHV-GT-F is present in the sequence of the genomic herpesvirus genome 15996-16015 or 288834-288853, and the KHV-GT-R is present in the sequence of the genome of the koi herpes virus 1614 1-16160 or 288979-288998. Complementary strand; If these KHV-GT primer sets are used, DNA containing a base sequence of 165 bp in length shown in SEQ ID NO: 6 can be amplified. If these KHV·TNFR bow 1 subgroups or KHV-GT primer sets are used, Beij can specifically amplify the 190 bp portion of the genomic herpesvirus genome 7689-7878 or 280527-280716, or the genome 15996, in a short time. The 165 bp portion of -16160 or 288834-288998 specifically detects the presence of the Koi herpes virus gene DNA in the test sample. The first primer or the second primer of the present invention can be produced by a usual DNA automatic synthesizer (for example, manufactured by Applied Biosystems), and by a well-known DNA synthesis method (e.g., a phosphonimide method). The koi herpesvirus detection method of the present invention using the probe for koi herpes virus detection of the present invention can be labeled by the koi herpes disease 119552.doc -15-200804604 毋 gene DN A in the sample to be tested The koi herpes virus detection probe is hybridized to detect the labeling substance bound to the probe; or directly

As the sample to be tested, a dNA extract extracted from a sample suspected of containing the Koi herpes virus by a conventional method can also be used, and a koi herpes virus gene DNA amplified by the pCR method can also be used.

Examples of the labeling substance include an enzyme, a fluorescent substance, a chemiluminescent substance, a radioactive isotope, biotin, avidin, and the like; specifically, a peroxidase (for example, horseradish peroxidase (h〇rseradish peroxidase) )), alkaline phosphatase, d-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, Αρο·glucose Enzymes such as oxidase, urease, luciferase or acetylcholinesterase, luciferin isothiocyanate, phycobilip (intrubin), rare earth metal chelate, dansyl chloride (1) (10) (iv) SUlf〇nyl Fluorescent substances such as Chl〇ride) or tetramethylrhodamine (10) ramethyl rhodamme is〇thi〇cyanate), radioisotopes such as 3h, 14匚, 125 or 131, biotin, avidin, or chemistry Luminescent substance. As the position of the present invention, if it contains the sequence of the sequence shown in SEQ ID NO: 5 or 6, or its complementary sequence; or the sequence of the sequence shown in SEQ ID NO: 8 is missing, substituted Or the addition of the test sequence 'and when used as a probe' can detect the DNA of the medicinal cancer virus; or under stringent conditions T, with the complementary sequence of the sequence comprising the sequence shown in SEQ ID NO: 5 or Hybridization, and when used as a probe, can detect the role of Jin (4) luxury virus, there is no particular limitation, the above-mentioned "1 or several tests \ · 119552.doc •16· 200804604 base deletion, substitution or addition of base "Base sequence" means, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, particularly preferably 1 to 5, any number of substituents deleted, substituted or The base sequence to be added. For example, DNA (mutant DNA) comprising the base sequence of one or several bases deleted, substituted or added may also be known by chemical synthesis, genetic engineering methods, induced mutations, and the like. Method of manufacture. Specifically, a method in which a DNA comprising a nucleotide sequence represented by SEQ ID NO: 5 or 6 is brought into contact with a drug which is a mutated source, a method of irradiating ultraviolet rays, a method of genetic engineering, or the like is used, and the DN A is used. Mutation DNA can be obtained by introducing a mutation. The site-specific mutation induction method, which is one of the genetic engineering methods, is a method for introducing a specific mutation at a specific position, and therefore, it is useful according to Molecular Cloning: A laboratory Mannual, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring. Harbor, NY·, 2001. (hereinafter, referred to as "Molecular Colonization 3rd Edition"), Current Protocols in Molecular Biology, Supplement 1 ~ 38, ^ John Wiley & Sons (1987-1997), etc. . The mutated DNA can be expressed by using an appropriate expression line, or a protein containing one or several amino acid-deleted, substituted or added amino acid sequences can be obtained. - The above-mentioned "base sequence for hybridization under stringent conditions" means using a nucleic acid such as DNA or RNA as a probe, and using a colony hybridization method, a plaque hybridization method, or a Southern blot hybridization method. The base sequence obtained can be specifically exemplified by using a filter for immobilizing DNA derived from colonies or plaques or a fragment of the DNA, at 0.7 to 1.0 119 119552.doc -17- 200804604

In the presence of NaCl, after hybridization at 65 ° C, use about 0.1 to 2 times the SSC solution (the composition of the 1 S concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate), at 65 ° C Under the conditions, the filter is cleaned and the DN A is identified. Hybridization can be carried out according to the methods disclosed in the third edition of Molecular Cloning. That is, under strict conditions, it means a condition in which so-called specific hybridization is formed without forming non-specific hybridization, and specifically, DNA having a homology of 50 to 70% or more is hybridized to each other, and homologous thereto. Lesser conditions • The conditions under which DNA does not hybridize with each other; or the usual Southern hybridization conditions, ie at a salt concentration equivalent to 65 ° C, IxSSC, 〇.i% SDS, or O.lxSSC, 0.1% SDS Conditions for hybridization. For example, DNA which can hybridize under stringent conditions includes DNA having a certain degree of homology with a base sequence of DNA used as a probe, and for example, it can be preferably exemplified as having 60% or more. More preferably, it is 70% or more, more preferably 80% or more, particularly preferably 90% or more, particularly preferably 95% or more, and most preferably φ is a homologous DNA of 98% or more. The method for obtaining the DNA of the present invention or the preparation method thereof is not particularly limited, and the KHV-TNFR primer set or the KHV-GT primer set disclosed in the present specification can be prepared, and the DNA gene of the Koi herpes virus (KHV) can be screened using the same. The library, whereby the target DN A is isolated, or the target DNA can be prepared by chemical synthesis according to a usual method. Hereinafter, the present invention will be more specifically described by way of examples, and the technical scope of the present invention is not limited to the examples. The experimental methods were carried out according to the methods disclosed in the third edition of Molecular Closing, unless otherwise disclosed. Also, 119552.doc -18- 200804604 uses the abbreviation for the following terms. CTAB/NaCl solution: 10% cetyltrimethylammonium oxalic acid solution containing 0.7M NaCl, Tris: trishydroxymethylaminomethane, Tris-hydrochloric acid: containing trimethylolamine methane, pH adjusted with hydrochloric acid Value, SDS · Sodium lauryl sulfide, EDTA: ethylenediaminetetraacetic acid, TBE buffer · Liquid containing 0.089 Tri Tris, 0.089 硼 boric acid, 0.002 Μ EDTA, BSA: bovine serum Albumin, ΤΈ: a solution containing 10 mM Tris-hydrochloric acid (pH 8.0), 1 mM EDTA (pH 8.0), dATP: deoxyadenosine triphosphate, dCTP: deoxycytidine triphosphate, dGTP: Deoxyguanosine triphosphate, dTTP: deoxythymidine triphosphate, Triton X-100: polyoxyethylidene (10) octylphenyl ether. Example 1 Determining KHV isolated from Koi in Japan, the United States, and Israel, ie, a sputum isolate (DDBJ registration number: AP008984), a US isolate (DDBJ registration number: DQ657948), and an Israeli isolate (DDBJ registration number: DQ177346) ) the entire genome sequence. Fig. 1 shows the total length (bp) of the KHV genomes and the predicted number of genes, and Fig. 2 shows the results of comparison of the homology of the KHV genomes. The following two sets of PCR primers were produced based on the KHV genome sequences. Further, the position of each primer in the genome of the Japanese isolate (KHV-J) is shown. 1. KHV-TNFR primer KHV-TNFR-F: 5f-acagtgtccgacttgtgcga_3, (genomic location··7689-7708, 280527-280546) KHV-TNFR-R: 5*-tggtgcccacatgtgcgttg-3? 119552.doc -19· 200804604 (Location of the genome: 7859-7878, 280697-280716) The KHV-TNFR primer was amplified to the following sequence of 190 bp. The underline indicates the primer sequence.

5,-ACAGTGTCCGACTTGTGCGACGTCTGCAACCCCTGCGACA

GGTTTGAGTACCTCTCACCAGCCGGCGTGTGCTGCAAGCCA

TGCTACCCTGGGTACTACGCCGTCCAGCACTGCGCCACTGC

CCACACAGCCAGCGTGTGTGAGGCCTGTCCAGTGGGCACCT ACAAGAGCAACGCACATGTGGGCACCA-3f

2. KHV-GT primer KHV-GT-F: 5f-gacactgaacatgaacactg-3f (genomic location: 15996-16015, 288834-288853) KHV-GT-R: S^gaatccatccccatcacccg-S1 (genomic location: 16141-16160, 288979-288998) Amplified by KHV-GT primer to the following sequence of 165 bp. The underline indicates the primer sequence.

5,-GACACTGAACATGAACACTGAACACAACAACATTCACAA

TATTCACAATAGCATTGTGTGTGTGTGTGTGTGTGTGTGTGT

GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGA

TAAGAGTATGGAATAATACTTACGGGTGATGGGGATGGATT C-3f The extraction of genomic DNA of KHV was carried out according to the third edition of molecular selection. The extracted DNA was used after dialysis against TE buffer. Example 2 The sequence existing at positions 7689 to 7708 of the KHV genome, i.e., 119552.doc -20-200804604, above KHV-TNFR-F, was used as an upstream primer, and similarly, it was present in the sequence of 7859 to 7787. The complementary strand, KHV-TNFR-R, was used as a downstream primer, and the KHV genome was used as a template to amplify the target DNA by PCR, and the base sequence of the obtained DNA was confirmed. At the time of amplification, Taq DNA polymerase manufactured by Takara Co., Ltd., and a 10-fold concentration reaction solution and a 10-fold concentration dNTP solution added to the enzyme were used as a 1-fold concentration. The reaction system was adjusted to 50 μl. This solution was placed in an automatic temperature adjuster, and PCR was carried out using the above PCR primer using the KHV genome as a template. The conditions of the PCR were carried out for 30 cycles at 95 ° C for 30 seconds, at 55 ° C for 30 seconds, and at 72 ° C for 30 seconds. After completion of the PCR reaction, the molecular weight of the amplified DNA fragment was measured by electrophoresis using a 0.8% agarose gel at 80 mA for 1 hour in TBE buffer. The detection of the amplified DNA fragment and the molecular weight were determined relative to the relative mobility of a commercially available 100 bp ladder. The DNA fragment was detected by staining the agarose gel in a TBE buffer containing ethidium bromide (1 pg/mL) and observing the fluorescence under ultraviolet light. The base sequence of the obtained DNA fragment was determined by the dideoxy method using the above heat-resistant DNA synthetase. As a result, as revealed in SEQ ID NO: 5 of the Sequence Listing, a DNA fragment having a base sequence of 190 bp in length was obtained. Example 3 The sequence of position 15996 to 16015 of the pathogen genome of KHV disease, that is, KHV_GT-F, was used as the upstream primer, and the complementary strand of the sequence existing in the 16141 to 16160, that is, KHV-GT. -R, as a downstream primer of 119552.doc -21-200804604, a gene which determines the pathogen type of KHV disease is amplified by PCR to determine the base sequence of the obtained DNA. In the same manner as in Example 1, a DNA fragment having a base sequence length of 165 bp as disclosed in SEQ ID NO: 6 of the Sequence Listing was obtained. Example 4 Using the computer analysis software (SDC software), the base sequences of the two DNA fragments obtained in Examples 2 and 3 were compared to whether or not they corresponded to a part of the base sequence of the KHV genome. At the same time, log on the database of gene sequences (http://www.iicbi.nih.gov/index.html) and use the computer analysis software (SDC software) to check whether there is a part with the same base sequence. Eukaryotic organisms and diseased Qin were compared and analyzed. As a result, it was confirmed that the base sequence of the obtained DNA fragment was present in the KHV genome, and it was also found from the results of the database that eukaryotes and viruses having the same base sequence portions as the DNA fragments did not exist. From the above, it can be considered that the obtained two DNA fragments of 190 bp and 165 bp in length are capable of specifically detecting the DNA sequence of KHV. Example 5 Using the primers used in Examples 2 and 3, and the reported primers for KHV detection, it was carried out by 曰 (J), the United States (A), Israel (Is), and Indonesia (In). The isolated KHV genome is a template for PCR, and whether the target DNA region can be amplified. The adjustment of the reagents for PCR, the test conditions, and the electrophoresis conditions were all the same as in Example 2. As the primer for KHV detection which has been reported, the modified Sph 1-5 primer set of Table 1 of the above Non-Patent Document 1 is used. 119552.doc -22· 200804604 The electrophoresis results of the PCR amplification products are shown in Fig. 1. The primers used in Examples 2 and 3 are the same as the already existing primers, and all of the KHVs obtained in the separation region are formed into clear DNA bands on the basis of electrophoresis, and the genomes are displayed. presence. Example 6 Using the primers used in Example 2 and Example 3 and the KHV detection primers which have been reported, the KHV genome isolated from Japan was subjected to PCR, and the detection sensitivity of KHV using each primer group was compared. Each PCR system was carried out in the same manner as in Example 2, and for the KHV genome, the amount of KHV genomic DNA mixed in each reaction solution was adjusted to 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, and 1 pg. Of the six, try to use PCR to amplify the genes used to determine the species. An electrophoresis photograph of the PCR amplification product is shown in Fig. 2. The primers used in Examples 2 and 3 are identical to the existing primers, and the molecular weight fractions obtained on the basis of electrophoresis respectively form a clear DNA band, but in the cited primers, the KHV genome is less than 100 pg. In contrast, the primer of the present invention showed a clear band generated by PCR amplification of DNA under the condition that the KHV genome was 10 pg. That is, it can be clarified that the primers obtained in the present invention all have the detection sensitivity of the KHV genome which has been reported to be 100 times as large as that of the guide, and the diagnostic technique of KHV disease has been dramatically improved. Example 7 Using the primers used in Example 2 and Example 3 and the KHV detection primers already reported, PCR was carried out on DNA extracted from three Koi carp infected with KHV, and KHV using each primer group was performed. Detection. 119552.doc -23- 200804604 Further, the control system used KHV genomic DNA isolated from Japan. Each PCR was carried out in the same manner as in Example 2. The extraction of DNA from the koi carp is carried out by the "DNA-Molecular Genetics Method of Fish" Chapter 2 Gene Analysis (Hirano Breeding), Ltd. Stellar Society Hosei Court (issued on June 10, 1997) The method disclosed is carried out. The electrophoresis results of the PCR amplification products are shown in Fig. 5. The primers used in Examples 2 and 3 all formed a KHV-specific DNA band on the basis of electrophoresis, and did not form a non-specific DNA band. In this regard, it has been reported that in the DNA of the three Koi scorpions infected with KHV, in addition to the KHV-specific DNA band, a non-specific DNA band is formed. That is, it is clear that the primers obtained in the present invention have superior detection accuracy to the KHV genome as compared with the already reported guides, and the diagnostic technique for KHV disease is dramatically improved. Industrial Applicability By using the probe group for koi herpes virus detection or the probe for koi blister virus detection of the present invention, the herpesvirus gene can be detected with high sensitivity, high precision, and rapidity. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the total length (bp) of the KHV genome of the Japanese isolate (KHV-J), the American isolate (KHV-A), the Israeli isolate (KHV-Ι), and the predicted number of genes. Figure. Fig. 2 is a graph showing the results of comparison of the homology of the KHV genome of the Japanese isolate (KHV-J), the US isolate (KHV-A), and the Israeli isolate (KHV-Ι). 119552.doc -24- 200804604 Fig. 3 is a view showing the results of electrophoresis of a PCR amplification product using the primer for KHV detection of the present invention. Fig. 4 is a graph showing the results of electrophoresis of PCR amplification products of the amount (6 types) of the KHV genome using the primer for detecting KHV pathogens of the present invention. Fig. 5 is a view showing the electrophoresis results of PCR amplification products of DNA of three Koi carp infected with KHV using the primer for detecting KHV pathogens of the present invention.

119552.doc -25- 200804604 Sequence Table &lt;110> National University Corporation Tokyo Ocean University &lt;120> Koi Herpes Virus (KHV) Detection Method &lt;130&gt; F2006-014 &lt;140&gt; 096112870 &lt;141&gt; 2007- 04-12 &lt;150> JP 2006-111415 <151 &gt; 2006-04-13 &lt;160&gt; 6 &lt;170&gt; Patent In3. 1 Edition

&lt;210> 1 &lt;211> 20 &lt;212> DNA &lt;213&gt;manual&lt;220&gt;&lt;223&gt; KHV - TN FR-F &lt;400> 1 acagtgtccg acttgtgcga &lt;210&gt; 2 &lt;211 〉 20 &lt;212> DNA &lt;213>Manual&lt;220〉

&lt;223>KH V — TN FR — R 10 &lt; 400〉 2 tggtgcccac atgtgcgttg &lt;210&gt; 3 &lt;211> 20 &lt;212> DNA &lt;213>manual&lt;220&gt;&lt;223&gt; KH V — GT - F &lt;400&gt; 3 gacactgaao atgaacactg 119552.doc 200804604 &lt;210&gt; 4 &lt;211&gt; 20 &lt;212> DNA &lt;213>manual&lt;220&gt;&lt;223>KHV - GT - R &lt;400&gt ; 4 gaatccatcc ccatcacacg &lt;210&gt; 5 &lt;211> 190 &lt;212> DNA &lt;213> Koi herpesvirus &lt;400> 5

acagtgtccg acttgtgcga cgtctgcaac ccctgcgaca ggtttgagta cctctcacca gccggcgtgt gctgcaagcc atgctaccct gggtactacg ccgtccagca ctgcgccact gcccacacag ccagcgtgtg tgaggcctgt ccagtgggca cctacaagag caacgcacat gtgggcacca &lt; 210> 6 &lt; 211 &gt; 165 &lt; 212> DNA &lt; 213> Kam key herpes virus &lt; 400 &gt; 6 gacactgaac atgaacactg aacacaacaa Cattcacaat attcacaata gcattgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtgt tgtgtgtgtgt tgtgtgtgtgt tgtgtgtgtg tgataagagt atggaataat acttacgggt gatggggatg gattc 2- 119552.doc

Claims (1)

200804604 X. Patent application scope: 1 · A primer group for detecting KHV in Jinyu fish, which is characterized in that it contains a first DNA primer and a second DNA primer, and the _Dna primer contains a sequence of the sequence table An oligonucleotide portion of at least one of the bases of the sequence of the sequence shown in No. 1, the second DNA primer comprising at least one base of the base sequence shown in SEQ ID NO: 2 of the sequence listing The acid part. 2. A primer set for detection of koi herpesvirus (KHV), characterized in that it comprises a first DNA primer and a second DNA primer, the first DNA primer comprising at least the sequence of the sequence number of sequence number 3 of the sequence listing An oligonucleotide portion of one of the bases, the second DNA primer comprising an oligonucleotide of at least one base of the base sequence of SEQ ID NO: 4 in the Sequence Listing. 3 · A method for detecting a spleen virus (KHV), characterized in that the basin is used as a kit for detecting koi herpesvirus (KHV) of claim 1 or 2 to amplify the koi in the sample to be tested Herpesvirus (KHV) gene. A probe for detecting koi herpesvirus (KHV), which comprises a nucleotide sequence of SEQ ID NO: 5 in the sequence listing or a nucleotide sequence of at least 10 bases complementary thereto section. 5. A probe for detecting a koi herpesvirus (KHV), characterized in that it contains a labeled nucleus of at least 10 bases of the nucleotide sequence shown in SEQ ID NO: 6 of the Sequence Listing or its complementary sequence. The glycoside moiety. 6. A method for detecting koi herpesvirus (KHV), characterized in that it is used under the condition that hybridization is possible, and the koi rash of 119552.doc 200804604 7. virus (KHV) is used as claimed in claim 4 or 5. The probe is cultured with the sample to be tested. A DN A comprising the base sequence shown in SEQ ID NO: 5 or 6, or its complementary sequence. A DNA comprising a base sequence in which one or several bases are deleted, substituted or added in a nucleotide sequence represented by SEQ ID NO: 5 or 6, and when used as a probe, can detect koi herpes Virus (KHV). A DNA which hybridizes under stringent conditions to DNA comprising a sequence complementary to the nucleotide sequence of SEQ ID NO: 5 or 6, and which, when used as a probe, detects koi herpesvirus (KHV). 119552.doc