Dual real-time fluorescent quantitative detection method for carp edema virus and koi herpes virus

Abstract

The invention discloses a dual real-time fluorescent quantitative PCR detection method for carp edema virus and koi herpes virus. The method includes the following steps: first, synthesizing primers and TaqMan probes: optimizing the primer sequence according to the CEV P4a gene sequence, The first base of the upstream primer was changed to a degenerate base W, and the first base of the downstream primer was changed to a degenerate base R; according to the conserved sequence of the KHV ORF7 gene, a pair of specific primers and a TaqMan probe were designed; Use FAM and VIC as the probe reporter group, and BHQ1 as the probe quencher group; second, determine the reaction system and conditions: use 20 μL reaction system, 2×Probe PCR Master Mix 10 μL, upstream and downstream primers and probes The final primer concentration was between 0.2 μmol / L and 0.8 μmol / L, QN ROX reference dye 0.1 μL, template 2.5 μL, and DEPC water to make up 20 μL; the reaction program was: 95°C pre-denaturation for 2 min, 1 cycle; 95°C for 5 s, Anneal at 50-60°C for 31s, 40 cycles.

Description

Technical field [0001] The present invention relates to a carp puffy virus and koi herpes virus dual real-time PCR detection method, belongs to the field of fish virus detection technology. Background [0002] Carp and koi are species with important market value in China, and with the continuous expansion of the scale of breeding, fish diseases also occur frequently. Among them, both carp edema virus (CEV) and koi herpes virus (Kioherpesvirus, KHV) can infect carp and koi, causing serious harm to the aquaculture industry. CEV is a linear double-stranded DNA that belongs to the pox virus, which is about 200 nm in size × 400 nm. KHV is also a double-stranded DNA, which belongs to the herpes virus, which is a icosahedron with a diameter of 167 to 200 nm and a capsule membrane. The above two viruses only infected carp and koi, and the clinical symptoms caused by them were extremely similar, including gills, eye depression, loss of appetite, body surface bleeding, etc., and the mortal...

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Problems solved by technology

There have been a large number of reports on sensitive cell lines against KHV, such as Koi fin cells (KFC), common carp brain cells (CCB), Koi gill cells (The gill of koi, KoG), etc. However, it usually needs to be passed 3 to 5 times, the isolation and culture time is longer, and the first isolation is rarely successful, so the ordinary PCR detection method is still the first choice for the diagnosis of KHV, but compared with the real-time fluorescent quantitative PCR method, it takes a long time. There are many detection steps

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