Specific primer, probe and kit for detecting Chinese soft-shelled turtle flavivirus
A Chinese soft-shelled turtle, flavivirus technology, applied in the direction of viruses, viral peptides, viruses/phages, etc., to achieve the effect of high mortality, long incubation period, and conducive to green sustainable development
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[0032] 1. Sample collection and virus purification
[0033] Take out the liver, spleen, lung, kidney and intestinal tissues of the diseased soft-shelled turtle, and homogenize them on ice. The homogenate was centrifuged at 12,000 g for 20 min, the supernatant was taken, filtered through a 0.22 μm bacterial filter, the filtrate was centrifuged at 40,000 g for 3 h, and the precipitate was resuspended in TNE buffer as the virus-containing crude extract. The above virus liquid was purified by discontinuous sucrose density gradient centrifugation.
[0034] 2. Viral nucleic acid extraction and high-throughput sequencing
[0035] Take 200 μL of the purified virus liquid, use the QIAamp MinElute Virus Spin Kit (QIAGEN Germany) kit to extract viral nucleic acid, and send it to Sangon Bioengineering (Shanghai) Co., Ltd. for RNA and DNA library construction, and then separately High-throughput sequencing, in which high-throughput sequencing of RNA library construction, splicing out par...
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