BVDV、BRV和BCoV TaqMan三重RT

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'-UTR、BRV NSP5和BCoV N基因扩增呈阳性，而对牛传染性鼻气管炎病毒、牛副流感病毒、魏氏梭菌（A型、B型和D型）、多杀性巴氏杆菌（A型和B型）等犊牛腹泻相关病原扩增均呈阴性；最低检出限为10拷贝/μL；组内和组间变异系数均小于2%。利用本研究建立的TaqMan三重RT-qPCR方法对29份临床样品进行检测，获得BVDV、BRV、BCoV的4种混合感染型，其中BVDV与BCoV的混合感染率最高（27.6%）；与已有单重RT-qPCR方法比较，发现两种方法检测BVDV、BRV和BCoV的符合率分别为100%、96.5%、100%。结果表明，本研究建立的TaqMan三重RT-qPCR方法具有特异性强、敏感性高、重复性好、可行性高等优点，可为今后BVDV、BRV和BCoV共感染引起的牛腹泻性疾病的鉴别诊断和流行病学调查提供新技术手段。

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 virus（IBRV），bovine parainfluenza virus（BPIV），Clostridium welchii（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.

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