In recent years,yellow catfish nidovirus(YCNdV)is a newly identified viral pathogen in cultured yellow catfish.To provide an accurate and sensitive detection method for YCNdV,this study designed specific primers and probes based on the capsid gene sequence of YCNdV.The reaction system and conditions were op-timized,a qPCR method was established along with a standard curve,and the qPCR method's sensitivity,speci-ficity,and stability were tested.The method was then applied to clinical samples,and its detection rate was com-pared with that of nested PCR.The results showed that the final optimized concentrations of the primers and probes were 0.4 μmol/L and 0.1 μmol/L,respectively,with the optimal annealing extension temperature being 58℃.Under these conditions,the minimum detection limit was 4 copies of the pMD19-YCNdV.cap plasmid standard.The generated standard curve demonstrated good linearity and a wide linear range(3.37×1010 copies/μL to 3.37×100 copies/μL),with a correlation coefficient(R2)of 0.998 4 and an amplification efficiency of 91.3%.Specificity testing revealed no cross-amplification signals with yellow catfish picornavirus(YCPrV),grass carp reovirus genotype Ⅱ(GCRV-Ⅱ),Cyprinid herpesvirus 2(CyHV-2),Micropterus salmoides rhab-dovirus(MSRV),largemouth bass virus(LMBV),infectious spleen and kidney necrosis virus(ISKNV),carp e-dema virus(CEV),and Koi herpesvirus(KHV).Reproducibility experiments showed that the coefficients of variation were less than 0.6%both between and within groups.Testing 32 stored suspected YCNdV infection samples revealed that the detection rate of TaqMan qPCR was 9.375%higher than that of nested PCR.Taq-Man qPCR detection of various tissues in diseased yellow catfish showed significantly higher viral loads of YC-NdV in the spleen,gills,and kidneys compared to other tissues.These results indicate that the TaqMan qPCR detection method established in this study possesses excellent specificity,reproducibility,and ultra-high sensitivi-ty,making it suitable for the quantitative detection of YCNdV.This method provides a reliable means for the early prevention and treatment of diseases caused by YCNdV.
Pelteobagrus fulvidraconidovirusTaqMan qPCRtissue distribution
维普
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