Dual real-time fluorescence quantitative detection method for CEV (carp edema virus) and KHV (Koi herpesvirus)

Abstract

The invention discloses a dual real-time fluorescence quantitative PCR (polymerase chain reaction) detection method for a CEV (carp edema virus) and a KHV (Koi herpesvirus). The method comprises following steps: step one, synthesis of primers and a TaqMan probe: a primer sequence is optimized according to a CEV P4a gene sequence, first base of an upstream primer is changed into degenerate base W,and first base of a downstream primer is changed into degenerate base R; a pair of specific primers and one TaqMan probe are designed according to KHV ORF7 gene conserved sequence; FAM and VIC are taken as probe reporter groups and BHQ1 is taken as a probe quenching group; step two, determination of a reaction system and conditions: a 20 mu L reaction system is adopted and determined as 10 mu L 2*Probe PCR Master Mix, final concentration of the upstream and downstream primers and the probe primers is in a range of 0.2 mu mol / L-0.8 mu mol / L, a QN ROX reference dye is 0.1 mu L, a template is 2.5mu L and the balance of DEPC water, and the total amount is 20 mu L; the reaction procedure is that predegeneration at 95 DEG C is performed for 2 min, and one cycle is executed; annealing is performed for 5 s at 95 DEG C and performed for 31 s at 50-60 DEG C, and 40 cycles are executed.

Description

technical field [0001] The invention relates to a dual real-time fluorescent quantitative PCR detection method for carp edema virus and koi herpes virus, belonging to the technical field of fish virus detection. Background technique [0002] Carp and koi are varieties with important market value in my country. With the continuous expansion of breeding scale, fish diseases also frequently appear. Among them, 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 belonging to poxviruses with a size of about 200nm×400nm. KHV is also double-stranded DNA, belongs to herpes virus, is an icosahedron with a diameter of 167-200nm, and has a capsule. The above two viruses only infect carp and koi, and the clinical symptoms caused are very similar, including gill rot, sunken eyeballs, loss of appetite, body surface bleeding, etc., and the mortality rate is as high...

AI Technical Summary

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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