爲建立牛病毒性腹瀉病毒（BVDV）務要、牛輪狀病毒（BRV）和牛冠狀病毒（弟請BCoV）的快速檢測方法，根據GenBank中登錄的BVDV 5'-U吧慢TR、BRV NSP5和BCoV N基因序列設計特異性引報通物和探針，通過(guò)優化反應也雜體系和條件，建立了同時(shí)檢測上述3種(zhǒng)病毒的TaqMa制懂n三重RT-qPCR方法。該方法僅對(duì)BVDV 5'-UTR不光、BRV NSP5和BCoV N基因擴增呈陽性，而對(duì)牛傳染性空門鼻氣管炎病毒、牛副流感病毒、魏氏梭菌（A型、B型和D型我海）、多殺性巴氏杆菌（A型和B型）等犢牛腹瀉相關病原擴增均呈陰性；最低檢出限爲1技校0拷貝/μL；組内和組間變異系數均小于2%。利用本研究建立的TaqMan三重R窗科T-qPCR方法對(duì)29份臨床樣(yàng)品進(j視事ìn)行檢測，獲得BVDV、B高資RV、BCoV的4種(zhǒng)混合感染型，其中B線好VDV與BCoV的混合感染率最高（27.6%）；與已有單重RT-qPCR方上校法比較，發(fā)現兩(liǎng)種(zhǒn校吧g)方法檢測BVDV、BRV和BCoV的符合率分别爲100%、兵兵96.5%、100%。結果表明，本研究建立的TaqMan三視少重RT-qPCR方法具有特異性強、敏感性高、重複性好(hǎ鐵劇o)、可行性高等優點，可爲今後(hòu)BVDV、BRV和B空開CoV共感染引起(qǐ)的牛腹瀉性疾病的鑒别診斷和流行病學(x愛她ué)調查提供新技術手段。

Establishment of TaqMan Tripl知匠e RT-qPCR for Detecting B哥很VDV，BRV and BCoV

In order to establish多腦 a rapid method for detecting bov子地ine viral diarrhea v睡業irus（BVDV），bovin光算e rotavirus（BRV）and bovine coron物少avirus（BCoV），the specific pri務知mers and probes 很上were designed accor如高ding to the sequence票冷s of 5'-UTR，BRV NSP5 and BCoV N要化 genes registered in GenBank，follo校從wed by the optimi湖關zation of reaction systems and condi劇綠tions，then the TaqMan tri日她ple RT-qPCR was establishe裡答d to simultaneously detect the a樂又bove three viruses.呢區 By which，positive resul紅做ts only occurred in the amp照請lification of BVDV 5'-UTR，BR嗎裡V NSP5 and BCoV N gene人信s，but negative for infect聽理ious bovine rhinotrachei開秒tis virus（IBRV），bovine parainf話快luenza virus（BPIV），Clostrid件說ium welchii（type 花兒A，B and D），Pasteurella multocida（type美動 A and B）and other pathoge看間ns related to calf dia少能rrhea；the minimum detection limit was 老匠10 copies/μL；and the va新員riation coefficients of intra-and inter又愛-group were less t志跳han 2%. 29 clinical sam體兵ples were detected by the est離購ablished method，the four types of c低這o-infection of BVDV，BRV and BCoV人是 were obtained，in which，the c風作o-infection rate of BVDV and BCoV房歌 was highest（up to 27.6%）. 一但The coincidence r能中ates of current single RT-qPCR and t外妹he established method for B慢麗VDV，BRV and BCoV were 1廠間00%，96.5% and 100%，respectiv綠物ely. As a conclusion，the established 道紙method could be applied in differenti能明al diagnosis and epidemio舞新logical investigation 好什against bovine di我通arrhoeal diseases ca開大used by the co-infection舞兒 with BVDV，BRV and BCoV in the future 和她due to its advantages他術 of good specificity，sensit市農ivity，repeatability and feasibi這文lity.

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