BVDV、BRV和BCoV TaqMan三重RT-qPCR检测方法的建立

为建立牛病毒性腹泻病毒(BVDV)、牛轮状病毒(BRV)和牛冠状病毒(BCoV)的快速检测方法,根据GenBank中登录的BVDV 5'-UTR、BRV NSP5和BCoV N基因序列设计特异性引物和探针,通过优化反应体系和条件,建立了同时检测上述3种病毒的TaqMan三重RT-qPCR方法.该方法仅对BVDV 5'-...

Establishment of TaqMan Triple RT-qPCR for Detecting BVDV,BRV and BCoV

In order to establish a rapid method for detecting bovine viral diarrhea virus(BVDV),bovine rotavirus(BRV)and bovine coronavirus(BCoV),the specific primers and probes were designed according to the sequences of 5'-UTR,BRV NSP5 and BCoV N genes registered in GenBank,followed by the optimization of reaction systems and conditions,then the TaqMan triple RT-qPCR was established to simultaneously detect the above three viruses.By which,positive results only occurred in the amplification of BVDV 5'-UTR,BRV NSP5 and BCoV N genes,but negative for infectious bovine rhinotracheitis viru(s IBRV),bovine parainfluenza viru(s BPIV),Clostridium welchi(i type A,B and D),Pasteurella multocida(type A and B)and other pathogens related to calf diarrhea;the minimum detection limit was 10 copies/μL;and the variation coefficients of intra-and inter-group were less than 2%.29 clinical samples were detected by the established method,the four types of co-infection of BVDV,BRV and BCoV were obtained,in which,the co-infection rate of BVDV and BCoV was highest(up to 27.6%).The coincidence rates of current single RT-qPCR and the established method for BVDV,BRV and BCoV were 100%,96.5%and 100%,respectively.As a conclusion,the established method could be applied in differential diagnosis and epidemiological investigation against bovine diarrhoeal diseases caused by the co-infection with BVDV,BRV and BCoV in the future due to its advantages of good specificity,sensitivity,repeatability and feasibility.