Establishment of a triplex RT-PCR assay for detection of bovine norovirus,bovine kobuvirus and bovine torovirus
In order to explore the prevalence of bovine norovirus(BNoV),bovine kobuvirus(BKV)and bovine torovirus(BToV)in China,three pairs of specific primers were designed according to the conserved regions of the RdRp genes of BNoV,the 3D genes of BKV and the N genes of BToV registered in GenBank.The triple RT-PCR method was successfully established for simultaneous and rapid detection of BNoV,BKV and BToV.This method has strong specificity,and only specific bands were amplified for BNoV,BKV and BToV,and no bands were amplified for other 5 common pathogens causing calf diarrhea.The sensitivity was high,and the minimum detection limits for pMD18-T-BNoV,pMD18-T-BKV and pMD18-T-BToV plasmid standards were 2.73 × 104 copies/μL,2.87 × 104 copies/μL and 3.18 × 103 copies/μL,respectively.The three test results of same batch samples consistently showed that the repeatability was good.A total of 153 clinical samples were detected by this method.The results showed that the positive detection rates of BNoV,BKV and BToV were 11.11％,25.49％and 38.56％,respectively.The positive mixed infection rate for all three viruses reached 7.18％.Compared with the single RT-PCR detection method established by other scholars,the coincidence rates for the three viruses mentioned above were 94.77％,93.46％and 91.50％,respectively.In this study,the triplex RT-PCR method provides technical support for rapid detection and epidemiological investigation of the above mentioned domestic emerging viruses in calves.
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