三种牛肠道病毒TaqMan荧光定量鉴别检测方法的建立OA

To establish a TaqMan real-time assay for differential detection of three bovine enteroviruses,the specific primers and probes were developed in accordance with the conserved gene sequences of bovine viral diarrhea virus(BVDV)and bovine coronavirus(BCoV),the concentrations of the primers and probes and the reaction parameters and conditions of the reaction system were optimized.The specificity,sensitivity and reproducibility tests were assessed.The method was compared with commercial kits to assess its practi-cal applicability.The results demonstrated that 55 ℃ served as the optimal annealing temperature,0.8 μL(10 μmol/L)and 0.3 μL(10 μmol/L)represented the optimal volume for primers and probes respectively in a 25.0 μL reaction system.The method could specifically detect BVDV,BCoV and BRV,and there was no cross-reaction with other non-target pathogens in cattle.The detection limit for BVDV,BCoV and BRV was 13.4 copies/μL,the intra-and inter-assay coefficient of variation were from 0.35％to 1.24％.The method and a commercial kit were used to simultaneously detect 346 clinical samples,and the positive coincidence rate of the two methods was from 96.97％to 100％.The results confirmed that the TaqMan Real-Time PCR assay for differential detection of BVDV,BCoV and BRV developed in this study was proven to be has strong specificity,high sensitivity and good reproducibility,which was helpful for the early differential di-agnosis,prevention and control of pathogens of digestive tract.