WO2017188631A1 - 고병원성 바이러스성 출혈성 패혈증 바이러스 검출용 바이오마커 및 진단방법 - Google Patents
고병원성 바이러스성 출혈성 패혈증 바이러스 검출용 바이오마커 및 진단방법 Download PDFInfo
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- the present invention relates to a biomarker composition for detecting a Viral Hemorrhagic Septicemia Virus (VHSV) and a method for diagnosing VHSV infection.
- VHSV Viral Hemorrhagic Septicemia Virus
- VHSV Viral Hemorrhagic Septicemia Virus
- VHS was first reported as an important viral disease of rainbow trout. Recently, various fish, trout, rainbow trout, coho salmon, river trout, brown trout and steelhead trout, as well as salmon and fish, such as cod and herring It is also widely used in marine farmed fish such as fish and halibut in the natural water system, and is mainly found in Europe, North America, and Asia.
- VHSV is a negative single-stranded RNA virus, first reported in 1963 at the World Animal Health Organization (OIE) Fish Bottle Symposium, bullet-shaped, 180 nm long, 60 nm in diameter, 11 kb in size and nucleocapsid protein.
- N nucleocapsid protein
- P polymerase-associated phosphoprotein
- M matrix protein
- G surface glycoprotein
- NV protein unique non-ionic protein
- NV protein which belongs to the rhabdovirus family consisting of six proteins, a unique non-virion protein (NV), and a virus polymerase (L), and among the six proteins that produce the VHSV. It has been reported to be related to the pathogenicity of this VHSV.
- VHSV gene mutations occur at a very rapid rate and new strains of the VHS virus continue to develop.
- the present invention is to provide a biomarker composition for detecting a high pathogenic viral hemorrhagic Septicemia virus (VHSV) to provide a method for early detection of new pathogenic VHSV and diagnosis of VHSV infection.
- VHSV viral hemorrhagic Septicemia virus
- the present invention is an amino acid sequence of the 56th amino acid serine substituted with leucine in the amino acid sequence of Viral Hemorrhagic Septicemia Virus (VHSV) consisting of the amino acid sequence represented by SEQ ID NO: 1; Amino acid wherein serine (S), the eighth amino acid, is substituted with asparagine (N); Amino acid where threonine (T), 81st amino acid, is substituted with alanine (A); An amino acid in which the 88th amino acid valine (V) is substituted with alanine (A); 5 to 5 or more selected from the group consisting of an amino acid in which 117th amino acid glycine (G) is substituted with aspartic acid (D) and an 119th amino acid glutamic acid (E) is substituted with lysine (K) It provides a biomarker composition for VHSV detection containing 120 consecutive polypeptides as an active ingredient.
- VHSV Viral Hemorrhagic Septicemia
- the present invention is a gene in which a viral hemorrhagic septicemia virus (VHSV) consisting of a nucleotide sequence represented by SEQ ID NO: 2, a gene in which the 23rd base of the sequence is replaced with adenosine (A); A gene in which the 167th base is substituted with thymine (T); A gene in which the 241th base is substituted with guanine (G); A gene in which the 262th base is substituted with adenosine (A); VHSV containing 5 to 360 consecutive polynucleotides selected as one or two or more selected from the group consisting of a gene in which the 350th base is substituted with adenosine (A) and a gene in which the 355th base is substituted with adenosine (A) It provides a biomarker composition for detection.
- VHSV viral hemorrhagic septicemia virus
- the present invention comprises the steps of sequencing the VHSV DNA sequence from a sample infected with Viral Hemorrhagic Septicemia Virus (VHSV); Converting the DNA sequence information obtained from the sequencing step into amino acid sequence information; And an amino acid in which serine (S), the 56th amino acid of the amino acid sequence information, is substituted with leucine (L); Amino acid wherein serine (S), the eighth amino acid, is substituted with asparagine (N); Amino acid where threonine (T), 81st amino acid, is substituted with alanine (A); An amino acid in which the 88th amino acid valine (V) is substituted with alanine (A); Detecting one or two or more from the group consisting of an amino acid substituted with aspartic acid (D) for glycine (G) as 117th amino acid and an amino acid substituted with lysine (K) for glutamic acid (E) as 119th amino acid; It provides a method for providing information
- the amino acid sequence of Viral Hemorrhagic Septicemia Virus consisting of the amino acid sequence represented by SEQ ID NO: 1 is a serine (S) amino acid substituted with leucine (L). ; Amino acid wherein serine (S), the eighth amino acid, is substituted with asparagine (N); Amino acid where threonine (T), 81st amino acid, is substituted with alanine (A); An amino acid in which the 88th amino acid valine (V) is substituted with alanine (A); An agent for detecting one or two or more from the group consisting of an amino acid in which the 117th amino acid glycine (G) is substituted with aspartic acid (D) and the 119th amino acid glutamic acid (E) is substituted with lysine (K) It provides a kit for detecting VHSV comprising.
- the 23rd base of the sequence is a gene substituted with adenosine (A); A gene in which the 167th base is substituted with thymine (T); A gene in which the 241th base is substituted with guanine (G); A gene in which the 262th base is substituted with adenosine (A);
- a VHSV detection kit comprising an agent for detecting one or more from the group consisting of a gene in which the 350th base is substituted with adenosine (A) and a gene in which the 355th base is substituted with adenosine (A).
- VHSV Viral Hemorrhagic Septicemia Virus
- the 56th amino acid of the six amino acid variants has been replaced with leucine. It was confirmed that high high pathogenicity was induced. Therefore, the six amino acid mutations can be used as a biomarker to detect new VHSV, and can be utilized as a biomarker for diagnosing viral hemorrhagic sepsis that can diagnose fish VHSV infection early.
- VHSV viral hemorrhagic sepsis virus
- Figure 2 is the result of converting the amplified DNA sequence of the NV protein region of viral hemorrhagic sepsis virus (VHSV) to the amino acid sequence and confirmed the mutated portion of the NV protein amino acid sequence.
- VHSV viral hemorrhagic sepsis virus
- VHSV viral hemorrhagic sepsis virus
- VHSV viral hemorrhagic sepsis virus
- FIG. 5 shows the pathogenicity of the NV amino acid variant proteins of six viral hemorrhagic sepsis viruses (VHSV). The result is confirmed.
- FIG. 6 is a result of confirming the hydropathy index of the NV variant protein of viral hemorrhagic sepsis virus (VHSV)
- Figure 6A is the result of confirming the change in the ATP production of the variant substituted with asparagine serine 8 amino acid
- Figure 6B is 56
- Figure 6C The result of confirming the change in ATP production of the mutant substituted with serine, leucine, the first amino acid
- Figure 6C The result of confirming the change in ATP production of the variant substituted with alanine, threonine, the 81st amino acid
- Figure 6D is valine
- the 88th amino acid (V) is the result of confirming the change in ATP production of the variant substituted with alanine (A).
- the present invention is an amino acid sequence of the 56th amino acid serine substituted with leucine in the amino acid sequence of Viral Hemorrhagic Septicemia Virus (VHSV) consisting of the amino acid sequence represented by SEQ ID NO: 1; Amino acid wherein serine (S), the eighth amino acid, is substituted with asparagine (N); Amino acid where threonine (T), 81st amino acid, is substituted with alanine (A); An amino acid in which the 88th amino acid valine (V) is substituted with alanine (A); 5 to 5 or more selected from the group consisting of an amino acid in which 117th amino acid glycine (G) is substituted with aspartic acid (D) and an 119th amino acid glutamic acid (E) is substituted with lysine (K)
- VHSV Viral Hemorrhagic Septicemia Virus
- VHSV viral hemorrhagic sepsis virus
- SEQ ID NO: 1 may be a unique non-virion protein (VNV) region that is a VHSV structural protein.
- VHSV Viral Hemorrhagic Septicemia Virus
- the present invention is a gene in which a viral hemorrhagic septicemia virus (VHSV) consisting of a nucleotide sequence represented by SEQ ID NO: 2, a gene in which the 23rd base of the sequence is replaced with adenosine (A); A gene in which the 167th base is substituted with thymine (T); A gene in which the 241th base is substituted with guanine (G); A gene in which the 262th base is substituted with adenosine (A); VHSV containing 5 to 360 consecutive polynucleotides selected as one or two or more selected from the group consisting of a gene in which the 350th base is substituted with adenosine (A) and a gene in which the 355th base is substituted with adenosine (A)
- VHSV viral hemorrhagic septicemia virus
- the present invention comprises the steps of sequencing the VHSV DNA sequence from a sample infected with Viral Hemorrhagic Septicemia Virus (VHSV); Converting the DNA sequence information obtained from the sequencing step into amino acid sequence information; And an amino acid in which serine (S), the 56th amino acid of the amino acid sequence information, is substituted with leucine (L); Amino acid wherein serine (S), the eighth amino acid, is substituted with asparagine (N); Amino acid where threonine (T), 81st amino acid, is substituted with alanine (A); An amino acid in which the 88th amino acid valine (V) is substituted with alanine (A); Detecting one or two or more from the group consisting of an amino acid substituted with aspartic acid (D) for glycine (G) as 117th amino acid and an amino acid substituted with lysine (K) for glutamic acid (E) as 119th amino acid; It provides a method for providing information
- the specimen may be selected from the group consisting of rockfish, flounder, dom, squid, mullet, perch, eel, flounder, puffer fish, mackerel, trout, larva, minnow, defense, horse mackerel, carp, smelt, eel, catfish, loach It may be, but may preferably be a flounder, but is not limited thereto.
- the sequencing in the sequencing step may use any method known in the art, but is not particularly limited thereto, using an automatic sequencer, pyrosequencing, PCR-RELP method (restriction) fragment length polymorphism (PCR), single strand conformation polymorphism (PCRSSCP), PCR-SSO (specific sequence oligonucleotide), allele specific oligonucleotide (ASO) hybridization using a combination of PCR-SSO and dot hybridization, TaqMan-PCR, MALDI- Any one or more selected from known methods such as TOF / MS, rolling circle amplification (RCA), high resolution melting (HRM), primer extension, Southern blot hybridization, and dot hybridization can be used.
- DNA sequence information obtained from the sequencing step may be a region of a unique non-virion protein (VNV) that is a VHSV structural protein.
- VNV unique non-virion protein
- the amino acid sequence of Viral Hemorrhagic Septicemia Virus consisting of the amino acid sequence represented by SEQ ID NO: 1 is a serine (S) amino acid substituted with leucine (L). ; Amino acid wherein serine (S), the eighth amino acid, is substituted with asparagine (N); Amino acid where threonine (T), 81st amino acid, is substituted with alanine (A); An amino acid in which the 88th amino acid valine (V) is substituted with alanine (A); An agent for detecting one or two or more from the group consisting of an amino acid in which the 117th amino acid glycine (G) is substituted with aspartic acid (D) and the 119th amino acid glutamic acid (E) is substituted with lysine (K) It can provide a kit for detecting VHSV comprising.
- the 23rd base of the sequence is a gene substituted with adenosine (A); A gene in which the 167th base is substituted with thymine (T); A gene in which the 241th base is substituted with guanine (G); A gene in which the 262th base is substituted with adenosine (A); It can provide a VHSV detection kit comprising an agent for detecting one or more than one in the group consisting of a gene 350th base is substituted with adenosine (A) and 355th base is a gene substituted with adenosine (A),
- the agent may be selected from the group consisting of primers and probes that specifically bind to the gene.
- the kit for detecting VHSV of the present invention includes primers or antibodies capable of selectively recognizing genes or proteins thereof in which base substitution has been performed, as well as tools and reagents generally used in the art for immunological analysis.
- Such tools or reagents include, but are not limited to, suitable carriers, labeling materials capable of generating detectable signals, solubilizers, detergents, buffers, stabilizers, and the like.
- the label is an enzyme, it may include a substrate and a reaction terminator that can measure the activity of the enzyme.
- Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art, such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyesters, Polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated with latex metal, other papers, glass, metals, agarose and combinations thereof.
- the detection kit of the present invention may preferably be an RT-PCR kit, a DNA kit or a protein chip kit.
- the RT-PCR kit may comprise individual primer pairs specific for the marker gene, as well as other test tubes or other suitable containers, reaction buffers (variable pH and magnesium concentrations), deoxynucleotides (dNTPs), Taq Enzymes such as polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC-water, sterile water and the like.
- reaction buffers variable pH and magnesium concentrations
- dNTPs deoxynucleotides
- Taq Enzymes such as polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC-water, sterile water and the like.
- the DNA chip kit may include a substrate on which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and a reagent, an agent, an enzyme, etc. for preparing a fluorescent probe, and the substrate may be a control gene. Or cDNA or oligonucleotide corresponding to fragments thereof.
- the protein chip kit may be a kit in which one or more antibodies against a marker are arranged at a predetermined position on a substrate and immobilized at a high density.
- the protein is separated from the sample, and the separated protein is hybridized with the protein chip to form an antigen-antibody complex, which can be read to confirm the presence or expression level of the protein.
- VHSV A viral hemorrhagic sepsis virus
- VHSV Viral Hemorrhagic Sepsis Virus
- VHSV viral hemorrhagic sepsis virus
- tissue pulverized product obtained after filtration was inoculated in EPC flounder cells at dilution ratios of 1: 100 and 1: 1000, and cultured at 16 ° C., and cell abnormalities were confirmed by microscopic observation.
- PCR primers Based on the gene sequence (SEQ ID NO: 2) of the VHSV NV protein, DNA polymerase chain reaction (PCR) primers (PCR) primers required for whole virus gene sequence analysis were prepared as shown in Table 2.
- PCR conditions were pre-denaturation at 95 °C for 5 minutes, 30 seconds denaturation at 95 °C, 30 seconds annealing at 58 °C, 30 seconds extension reaction at 72 °C 1 cycle, 25 cycles were reacted.
- the amplification product was electrophoresed on a 1% agarose gel to which ethidium bromide was added, using 1 ⁇ buffer as a buffer for electrophoresis, and the band was observed by a UV detector.
- PCR amplification products were collected using a gel purification kit (QIAGEN, Germany), and then each sample was mixed 1: 1 and used as a template, and PCR was performed once again under the above conditions using an NV ORF primer. After electrophoresis after PCR, DNA bands were observed by UV detector. PCR amplification products were recovered using a gel purification kit (QIAGEN, Germany), and cloned using TOPcloner TM TAcoreKit (Enzynomics, Korea) to perform sequencing.
- the DNA product obtained after the PCR reaction was confirmed using an agarose gel to obtain a DNA fragment as shown in FIG. 1.
- the ORF sequence of the VHSV NV gene was translated to obtain an amino acid sequence.
- the amino acid sequence was aligned to identify the sequence changed due to mutation. All the above process was performed using the Bioedit program.
- VHSV high pathogenicity of VHSV was confirmed by analyzing the production of ATP, a cellular energy in the flounder cells.
- a recombinant vector was constructed by cloning the wild type VHSV NV gene into a pcDNA3 vector.
- the recombinant vector thus prepared was transduced into HINAE cells (National Institute of Fisheries Science), which are embryonic cells of olive flounder, to express VHSV NV protein. 24 hours after transduction, ATP production, a cell energy, was measured according to the manufacturer's instructions using the ATP Bioluminescene Assay Kit (Roche, Switzerland) to confirm changes in cell energy generation according to VHSV NV protein expression.
- VHSV Viral hemorrhagic sepsis virus
- the base sequence of the six amino acid mutation sites disclosed in Table 3 was cloned into a pcDNA3 vector to prepare a recombinant vector.
- the prepared vector was transduced into the flounder cells in the same manner as in Example 2-1, and each of the six amino acid variants (No. 8, S ⁇ N; No. 56, S ⁇ L; No. 81, T) in the cell ⁇ A; No. 88, V ⁇ I; No. 117, G ⁇ D and No. 119, E ⁇ K) and then ATP production rate was analyzed.
- ATP production was reduced by up to 45% compared to wild-type VHSV NV protein, and among the 6 variants, No.
- the 56 (serine to leucine) amino acid variant showed the greatest reduction.
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Abstract
Description
이름 | 지역 | 연도 |
VHSV-KR-JJ-2 | 제주 | 2012 |
VHSV-KR-JJ-3 | 제주 | 2012 |
VHSV-KR-JJ-4 | 제주 | 2012 |
VHSV-KR-JJ-5 | 제주 | 2012 |
VHSV-KR-JJ-6 | 제주 | 2012 |
VHSV-KR-JJ-7 | 제주 | 2013 |
VHSV-KR-JJ-8 | 제주 | 2013 |
VHSV-KR-JJ-9 | 제주 | 2013 |
VHSV-KR-JJ-10 | 제주 | 2013 |
VHSV-KR-JJ-12 | 제주 | 2014 |
VHSV-KR-JJ-13 | 제주 | 2014 |
VHSV-KR-JJ-14 | 제주 | 2014 |
VHSV-KR-PH | 포항 | 2013 |
프라이머 | 염기서열 | 위치 | 크기 |
VHSV NO 1-F | GAACTCAGTTGAAAAATGGAAGGG 24bp | 152-175 | 1877 |
VHSV NO 1-R | CAACTTGAACTTCTTCATGGC 21bp | 2028-2008 | |
VHSV NO 2-F | TCGGACAACTCCTAAGACGTA 21bp | 1733-1753 | 2272 |
VHSV NO 2-R | CGGGTGACTAGGACGAAACTT 21bp | 4004-3984 | |
VHSV NO 3-F | ATCTCATTACCAACATGGCTCAAA 24bp | 3892-3915 | 2040 |
VHSV NO 3-R | TTGTTCGCTTCTCCCCTAATTGT 23bp | 5931-5909 | |
VHSV NO 4-F | TGCCATAGACCTACTCAAGTTAT 23bp | 5812-5834 | 2230 |
VHSV NO 4-R | CTGATCCATGGTGGCTATGTGAT 23bp | 8041-8019 |
염기서열 변이부위 | 염기서열 | 아미노산 변이부위 | Tm | GC | 서열번호 |
point mutation 23(G → A) | CGGCACACAACACAACCAGC 2Obp | NO.8(S → N) | 59.5℃ | 60.00% | 3 |
point mutation 167(C → T) | CTAGAGTCTTAGAGGATCTAAGGAC 25bp | NO.56(S → L) | 58.9℃ | 44.00% | 4 |
point mutation 241(A → G) | GTCTCCTAGAGGGAGCTCATTAC 23bp | NO.81(T → A) | 60.2℃ | 52.17% | 5 |
point mutation 262(G → A) | CTAAGGAATATCCCCTCCAGTCC 23bp | NO.88(V → I) | 60.2℃ | 52.17% | 6 |
point mutation 350(G → A) | GACGAATGACTCCGAATCTCCC 22bp | NO.117(G → D) | 60.0℃ | 54.55% | 7 |
point mutation 355(G → A) | GACGAATGGCTCCAAATCTCCC 22bp | NO.119(E → K) | 58.1℃ | 50.00% | 8 |
Claims (9)
- 서열번호 1로 표시되는 아미노산 서열로 이루어진 바이러스성 출혈성 패혈증 바이러스(Viral Hemorrhagic Septicemia Virus; VHSV)의 아미노산 서열 내에서, 56번째 아미노산인 세린(S)이 루이신(L)으로 치환된 아미노산; 8번째 아미노산인 세린(S)이 아스파라긴(N)으로 치환된 아미노산; 81번째 아미노산인 트레오닌(T)이 알라닌(A)로 치환된 아미노산; 88번째 아미노산인 발린(V)이 알라닌(A)으로 치환된 아미노산; 117번째 아미노산인 글리신(G)이 아스파르트산(D)으로 치환된 아미노산 및 119번째 아미노산인 글루타믹산(E)이 라이신(K)으로 치환된 아미노산으로 이루어진 군에서 하나 또는 둘 이상 선택되는 5 내지 120개의 연속적인 폴리펩티드를 유효성분으로 함유하는 VHSV 검출용 바이오마커 조성물.
- 청구항 1에 있어서, 상기 서열번호 1로 표시되는 바이러스성 출혈성 패혈증 바이러스(VHSV)의 아미노산 서열은 VHSV 구조단백질인 비리온성 단백질 (a unique non-virion protein; NV) 영역인 것을 특징으로 하는 고병원성 VHSV 검출용 바이오마커 조성물.
- 서열번호 2로 표시되는 염기서열로 이루어진 바이러스성 출혈성 패혈증 바이러스(Viral Hemorrhagic Septicemia Virus; VHSV)의 유전자 내에서, 상기 서열의 23번째 염기가 아데노신(A)으로 치환된 유전자; 167번째 염기가 티민(T)으로 치환된 유전자; 241번째 염기가 구아닌(G)으로 치환된 유전자; 262번째 염기가 아데노신(A)으로 치환된 유전자; 350번째 염기가 아데노신(A)으로 치환된 유전자 및 355번째 염기가 아데노신(A)으로 치환된 유전자로 이루어진 군에서 하나 또는 둘 이상 선택되는 5 내지 360개의 연속적인 폴리뉴클레오티드를 유효성분으로 함유하는 VHSV 검출용 바이오마커 조성물.
- 바이러스성 출혈성 패혈증 바이러스(Viral Hemorrhagic Septicemia Virus; VHSV)에 감염된 검체로부터 VHSV DNA 염기서열을 시퀸싱하는 단계;상기 시퀀싱 단계로부터 얻어진 DNA 염기서열 정보를 아미노산 서열 정보로 변환하는 단계; 및상기 아미노산 서열 정보 중 56번째 아미노산인 세린(S)이 루이신(L)으로 치환된 아미노산; 8번째 아미노산인 세린(S)이 아스파라긴(N)으로 치환된 아미노산; 81번째 아미노산인 트레오닌(T)이 알라닌(A)로 치환된 아미노산; 88번째 아미노산인 발린(V)이 알라닌(A)으로 치환된 아미노산; 117번째 아미노산인 글리신(G)이 아스파르트산(D)으로 치환된 아미노산 및 119번째 아미노산인 글루타믹산(E)이 라이신(K)으로 치환된 아미노산으로 이루어진 군에서 하나 또는 둘 이상 검출하는 단계를 포함하는 VHSV 감염 진단에 필요한 정보를 제공하는 방법.
- 청구항 4에 있어서, 상기 검체는 우럭, 넙치, 돔, 능성어, 숭어, 농어, 전어, 가자미, 복어, 고등어, 노래미, 다랑어, 민어, 방어, 전갱이, 잉어, 향어, 뱀장어, 메기, 미꾸라지 및 붕어로 이루어진 군에서 선택되는 것을 특징으로 하는 VHSV 감염 진단에 필요한 정보를 제공하는 방법.
- 청구항 4에 있어서, 상기 시퀀싱 단계로부터 얻어진 DNA 염기서열 정보는 VHSV 구조 단백질인 비리온성 단백질 (a unique non-virion protein; NV) 영역인 것을 특징으로 하는 VHSV 감염 진단에 필요한 정보를 제공하는 방법.
- 서열번호 1로 표시되는 아미노산 서열로 이루어진 바이러스성 출혈성 패혈증 바이러스(Viral Hemorrhagic Septicemia Virus; VHSV)의 아미노산 서열 내에서, 56번째 아미노산인 세린(S)이 루이신(L)으로 치환된 아미노산; 8번째 아미노산인 세린(S)이 아스파라긴(N)으로 치환된 아미노산; 81번째 아미노산인 트레오닌(T)이 알라닌(A)로 치환된 아미노산; 88번째 아미노산인 발린(V)이 알라닌(A)으로 치환된 아미노산; 117번째 아미노산인 글리신(G)이 아스파르트산(D)으로 치환된 아미노산 및 119번째 아미노산인 글루타믹산(E)이 라이신(K)으로 치환된 아미노산으로 이루어진 군에서 하나 또는 둘 이상을 검출하는 제제를 포함하는 VHSV 검출용 키트.
- 서열번호 2로 표시되는 염기서열로 이루어진 바이러스성 출혈성 패혈증 바이러스(Viral Hemorrhagic Septicemia Virus; VHSV)의 유전자 내에서, 상기 서열의 23번째 염기가 아데노신(A)으로 치환된 유전자; 167번째 염기가 티민(T)으로 치환된 유전자; 241번째 염기가 구아닌(G)으로 치환된 유전자; 262번째 염기가 아데노신(A)으로 치환된 유전자; 350번째 염기가 아데노신(A)으로 치환된 유전자 및 355번째 염기가 아데노신(A)으로 치환된 유전자로 이루어진 군에서 하나 또는 둘 이상을 검출하는 제제를 포함하는 VHSV 검출용 키트.
- 청구항 8에 있어서, 상기 제제는 상기 유전자에 특이적으로 결합하는 프라이머 및 프로브로 이루어진 군에서 선택되는 것을 특징으로 하는 VHSV 검출용 키트.
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