Fluorescent quantitative PCR detection kit for infectious spleen and kidney necrosis virus and Fluorescent quantitative PCR detection method of infectious spleen and kidney necrosis virus

A spleen and kidney necrosis virus, detection kit technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, microbial determination/inspection, etc., to achieve the effect of saving costs and simplifying operating procedures

Active Publication Date: 2014-08-06
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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However, there is no report on the application of fluorescence quantitative PCR

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  • Fluorescent quantitative PCR detection kit for infectious spleen and kidney necrosis virus and Fluorescent quantitative PCR detection method of infectious spleen and kidney necrosis virus
  • Fluorescent quantitative PCR detection kit for infectious spleen and kidney necrosis virus and Fluorescent quantitative PCR detection method of infectious spleen and kidney necrosis virus
  • Fluorescent quantitative PCR detection kit for infectious spleen and kidney necrosis virus and Fluorescent quantitative PCR detection method of infectious spleen and kidney necrosis virus

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Embodiment

[0038] 1. Design of specific primers and probes

[0039] According to the ORF007 protein nucleic acid sequence (NC_003494) retrieved from Genbank, primers and probes were designed for the detection of Siniperca ISPKV in the conserved gene fragment by blast comparison. The sequence is as follows:

[0040] ISKNV - F: 5'-CGAGGCCACATCCAACATC-3' (SEQ ID NO: 1);

[0041] ISKNV - R: 5'-CGCCTTTAACGTGGGATATATTG-3' (SEQ ID NO: 2);

[0042] ISKNV-P: 5'-FAM-CACCAAACTGACCGCGGACTCGT-Eclipse-3' (SEQ ID NO: 3);

[0043] The fluorescent probe reporter group is FAM, and the quencher group is Eclipse.

[0044] 2. Preparation of positive standard

[0045] Extract the nucleic acid sequence of mandarin fish infectious spleen and kidney necrosis virus ORF007 protein, and insert it into the T vector by conventional methods, which is the positive standard.

[0046] 3. Extraction of viral DNA

[0047] According to the instructions of Qiagen Blood Tissue DNA Extraction Kit (purchased from Qiagen, ...

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Abstract

The invention discloses a fluorescent quantitative PCR detection kit for infectious spleen and kidney necrosis viruses and a fluorescent quantitative PCR detection method of the infectious spleen and kidney necrosis viruses. The detection kit and the detection method are characterized in that a specific primer and a probe are designed according to the sequence of a gene conservative area of the infectious spleen and kidney necrosis virus ORF007. The detection kit and the detection method have the advantages that the fluorescent quantitative PCR technology is adopted, the detection is simple, convenient and quick, the sensitivity is high, the specificity is strong, the repeatability is good, and the detection kit and the detection method can be applicable to monitoring of the infectious spleen and kidney necrosis viruses, and can also be used for detecting the virus content and the virus titer in siniperca chuatsi.

Description

technical field [0001] The invention relates to a fluorescent quantitative PCR detection kit and a detection method, in particular to a fluorescent quantitative PCR detection kit and detection method of mandarin fish infectious spleen and kidney necrosis virus. Background technique [0002] Mandarin fish (Siniperca chuatsi) is one of the important freshwater fish species in my country. The iridescent virus disease of mandarin fish caused by infectious spleen and kidney necrosis virus (ISKNV) is the main disease of mandarin fish culture. Cause the mortality of mandarin fish to be close to 100%, become the main bottleneck restricting the development of mandarin fish aquaculture. In 1997, Wu Shuqin and others first discovered a spherical virus with a diameter of 150nm in sick mandarin fish, and considered it to be the pathogen of the fulminant infectious disease of mandarin fish. He Jianguo and others further proved the pathogenicity of the virus through reinfection. Through hi...

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/70C12Q2531/113C12Q2545/114C12Q2563/107
Inventor 林强付小哲李宁求吴淑勤石存斌
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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