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A rapid test kit and detection method for the inactivation of mandarin fish 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, microorganism determination/inspection, etc., to achieve the effect of shortening production cycle and improving production efficiency

Active Publication Date: 2019-11-19
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

However, there is no report on the rapid detection method for the inactivation of the mandarin fish Infectious Spleen and Kidney Necrosis Virus (ISKNV).

Method used

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  • A rapid test kit and detection method for the inactivation of mandarin fish infectious spleen and kidney necrosis virus
  • A rapid test kit and detection method for the inactivation of mandarin fish infectious spleen and kidney necrosis virus
  • A rapid test kit and detection method for the inactivation of mandarin fish infectious spleen and kidney necrosis virus

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Embodiment

[0027] 1. Screening of target genes

[0028] According to the transcription profile results of ISKNV-infected CPB cells in our laboratory, five viral ORFs with the highest expression levels were selected, and Primer 5.0 software was used to design specific primers and probes (see Table 1). ISKNV was serially diluted to 1, 10, 100, and 1000 copies / mL and inoculated into T25 cell culture flasks respectively. Seven days after inoculation, total RNA was extracted and reverse-transcribed to prepare cDNA templates. Using Premix Ex Taq TM (Probe qPCR) kit was used to detect the expression levels of these genes. Reaction system: Add 10 μL of Mix, 0.4 μL of ROX, 0.4 μL of each 10 μmol / L primer ISKNV FP / ISKNV RP, 0.4 μL of 10 μmol / L probe ISKNV probe, 2 μL template, and replenish water to 20 μL. In the negative control group, the samples to be tested were replaced with deionized water. The reaction conditions were: 95°C for 30s, 1 cycle; 95°C for 5s, 60°C for 34s, 40 cycles, and the ...

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Abstract

The invention discloses a siniperca chuatsi infectious spleen and kidney necrosis virus (ISKNV) inactivation rapid detection kit and a detection method. The detection kit and the detection method can monitor weather ISKNV is inactivated through detection of mRNA virus, specific steps are as follows: 7 days after an inactivated vaccine semi-finished product or finished product is inoculated with CPB cells, total cellular RNA is extracted, DNA residues are removed, reverse transcription is performed, a specific primer and a specific probe designed according to a siniperca chuatsi infectious spleen and kidney necrosis virus (ISKNV) ORF099 gene conservation region sequence are used for fluorescence quantitative PCR reaction, and weather the ISKNV is completely inactivated can be determined according to a reaction result. The detection kit and the detection method can be used for rapid detection of siniperca chuatsi infectious spleen and kidney necrosis virus, can simplify inactivation inspection operation procedures of the ISKNV cell inactivated vaccine, shortens the test cycle, saves the cost, improves the sensitivity of the inactivation inspection, and improves vaccine production efficiency.

Description

technical field [0001] The invention relates to a rapid detection kit and detection method for infectious spleen and kidney necrosis virus inactivation, in particular to a fluorescent quantitative RT-PCR detection kit and detection method for rapid detection of ISKNV inactivation based on viral mRNA monitoring. Background technique [0002] Mandarin fish (Siniperca chuatsi) is an important species of high-quality freshwater fish consumption and export earnings in my country. It is deeply loved by consumers because of its delicious taste and high protein content. However, serious disease problems have become the main bottleneck restricting the development of mandarin fish aquaculture. Infectious spleen and kidney necrosis virus (ISKNV) in mandarin fish has the characteristics of high morbidity, high pathogenicity, and large economic losses. The research on its prevention and control technology has become one of the important topics for fish disease researchers. Vaccination i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2563/107C12Q2521/107
Inventor 李宁求付小哲张醴溪林强刘礼辉梁红茹黄志斌
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI