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LAMP detection method of mandarin fish infectious spleen and kidney necrosis viruses

A technology of spleen and kidney necrosis virus and detection method, which is applied in the field of LAMP detection of mandarin fish infectious spleen and kidney necrosis virus, can solve problems such as being unsuitable for clinical large-scale detection, and achieve the effects of low cost, high amplification efficiency and simple operation.

Inactive Publication Date: 2014-07-30
HUAZHONG AGRI UNIV
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Problems solved by technology

[0003] At present, for the detection of ISKNV, there are mainly histological detection methods and molecular biological technology detection methods, but in actual operation, due to the limitation of their own conditions, these detection methods are not suitable for clinical mass detection

Method used

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  • LAMP detection method of mandarin fish infectious spleen and kidney necrosis viruses
  • LAMP detection method of mandarin fish infectious spleen and kidney necrosis viruses
  • LAMP detection method of mandarin fish infectious spleen and kidney necrosis viruses

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Embodiment 1

[0028] 1. Design and synthesis of LAMP primers:

[0029] The present invention utilizes the BLAST software in NCBI to carry out sequence analysis on the whole genome of ISKNV in GeneBank, and find out the specific conserved region ORF72 gene ( figure 1 As shown), 4 specific primers were designed for the 6 regions of the specific conserved region, and multiple sets of primers were designed using the online software of Eiken’s patented LAMP primerexplorer (http: / / primerexplorer.jp / e / index.html). Each set of primers includes a pair of F3 / B3 inner primers and a pair of FIP / BIP outer primers; and LAMP is used to screen the designed specific primers to determine highly specific primers.

[0030] The nucleotide sequences of specific primers are as follows:

[0031] ISKNV F3 5`-GCCTGGAAGACATACCCGA-3`

[0032] ISNNV B3 5`-CGTCATGTACGCGGACATG-3`

[0033] ISKNV FIP 5`-CGCAGGCAATCCATAAGCGCTTTTCATACCGCCGGAATCTTACC-3`

[0034] ISKNV BIP 5`-CCGGAGCCGTGCGCAATAAATTTTCGAGGAACGACACGTACTG-3` ...

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Abstract

The invention discloses a LAMP detection method of mandarin fish infectious spleen and kidney necrosis viruses. The LAMP detection method comprises the following steps of designing four specific primers according to six areas of an infectious spleen and kidney necrosis virus specific segment, carrying out fast amplification on the template DNA specific area by LAMP, and adding a dye and observing a color change under natural light by naked eyes thereby determining a result. The LAMP detection method has the whole reaction time less than or equal to 2h and LAMP forms waterfall-type amplification so that amplification efficiency is very high, sensitivity is improved by 100 times than that of common PCR, a complex detection apparatus and a detection process are avoided, processes are simple and on-site real-time detection is realized. Therefore, the LAMP detection method of mandarin fish infectious spleen and kidney necrosis viruses has the advantages of fast detection, high sensitivity, simple and convenient operation, low cost and easy determination of a detection result.

Description

technical field [0001] The invention relates to a virus detection method, in particular to a LAMP detection method for mandarin fish infectious spleen and kidney necrosis virus. Background technique [0002] Infectious spleen and kidney necrosis virus (ISKNV) was isolated from mandarin fish in 1998. The virus has an envelope and a diameter of 150nm. It is a cytoplasmic double-stranded linear DNA virus and is the main pathogen of the fulminant infectious disease of mandarin fish. It is mainly prevalent in mandarin fish cultured in freshwater in southern my country. The virus has a fast epidemic and a high incidence , and there are no effective control measures at present, seriously affecting the development of mandarin fish aquaculture. He Jianguo and others proved the pathogenicity of the virus through recurrent infection, thought it might be an iridescent virus, and conducted research on the tissue range of its infection, and found that the spleen and kidney were the main i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6844C12Q1/70C12Q2531/119C12Q2563/107
Inventor 刘学芹刘广鑫林蠡袁军法李泽明柳力月罗培骁
Owner HUAZHONG AGRI UNIV
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