Establishment and application of a one-step multiple fluorescence quantitative PCR method for detection of BCoV,BEV and BVDV
To establish a rapid real-time fluorescence PCR method for the detection of bovine coro-navirus(BCoV),bovine enterovirus(BEV),and bovine viral diarrhea virus(BVDV)in clinical diagnostics,three sets of specific primers and probes were designed based on the BCoV N gene,BEV 5'-UTR gene,and BVDV 5'-UTR gene.Through optimization of primer and probe concentrations,as well as reaction condi-tions,a one-step triplex real-time fluorescence PCR method was developed for simultaneous detection of bovine coronavirus,bovine enterovirus,and bovine viral diarrhea virus.The sensitivity,repro-ducibility,specificity,and clinical sample detection results of this triplex PCR method were as-sessed.The results demonstrated high sensitivity,with the minimum detection limits for BCoV,BEV,and BVDV being 10 copies/μL.Reproducibility was stable,with intra-batch and inter-batch coefficients of variation(CV)both below 2％.The method exhi-bited strong specificity,showing no cross-reaction with common diarrheal pathogens.This method detected positive rates of 6.5％(29/445)、17.5％(78/445)and 12.1％(54/445),respectively,in 445 bovine diarrhea fecal samples from Henan Province.These findings indicate the successful establishment of a one-step triplex real-time fluorescence PCR method for BCoV,BEV,and BVD-V,providing an effective technical approach for the rapid detection and prevention of these diseases.
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