In order to establish a rapid and sensitive TaqMan qPCR method for the detection of Micropterus salmoides rhabdovirus(MSRV),in this study,NCBI was used to analyze the conserved sequences of N genes of MSRV epidemic strains.The correct constructed plasmid standard pMD18-MSRV-N was identified by PCR and sequencing.The TaqMan qPCR method for MSRV was preliminarily established by optimizing various conditions.Results showed that the R2 of the standard curve of the established TaqMan qPCR method was 0.9906,and the plasmid standard had a good linear relationship with the respective Ct values within the range of 6.79×108 copies/μL to 6.79×102 copies/μL.The specificity of the method was evaluated by detecting different viruses.This method can specifically detect MSRV.However,the test results of eight common fish viruses were negative,including grass carp reovirus(GCRV),nervous necrosis virus(NNV),spring viremia of carp virus(SVCV),largemouth bass birnavirus(LBBV),Siniperca chuatsi iridovirus,infectious spleen and kidney necrosis virus(ISKNV),singapore grouper iridovirus(SGIV)and largemouth bass ranavirus(LMBV).The sensitivity of the method was evaluated by detecting different concentrations of plasmid standards(6.79×108 copies/μL to 6.79×101 copies/μL).Results showed that the detectable limit of the method was 6.79×102 copies/μL,which was 1000 times more sensitive than conventional PCR methods reported in the literature.The intra-assay and inter-assay repeatability of the method were evaluated by using different batches or different concentrations of plasmid standards extracted from the same batch as templates.The results showed that the coefficient of variation of the intra-assay and inter-assay repeatability of the method was less than 2%.The TaqMan qPCR and conventional PCR methods were used to detect seventy-one tissue samples from diseased Micropterus salmoides,Siniperca chuatsi,and dace.The results showed that the positive rate was 16.9%(12/71),and the negative detection rate was 83.1%(59/71).All the positive samples were detected from the diseased Micropterus salmoides,but no virus was detected from the diseased siniperca chuatsi or dace.The positive rate of the conventional PCR method was 9.9%(7/71),the negative sample rate was 90.1%(64/71),and the total coincidence rate of the two methods was 93.0%.The TaqMan qPCR method for MSRV established in this study has high specificity,sensitivity,repeatability,and accuracy.It can be used to detect clinical samples of Micropterus salmoides,which provides a feasible technical means for the rapid detection of MSRV.
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