牛病毒性腹泻病毒和牛冠状病毒双重纳米RT-PCR检测方法的建立及应用  

Establishment and application of a dual nano RT-PCR for the detections of bovine viral diarrhea virus and bovine coronavirus

作　　者：魏宇 王海璐 孙飞雁 郭利[2] 程悦宁[2] 王全凯[1] WEI Yu;WANG Hailu;SUN Feiyan;GUO Li;CHENG Yuening;WANG Quankai(College of Chinese Herbal Medicine,Jilin Agricultural University,Changchun 130112,China;Institute of Special Animal and Plant Science,Chinese Academy of Agricultural Sciences,Changchun 130112,China)

机构地区：[1]吉林农业大学中药材学院,吉林长春130112 [2]中国农业科学院特产研究所,吉林长春130112

出　　处：《畜牧与兽医》2022年第3期101-105,共5页Animal Husbandry & Veterinary Medicine

基　　金：吉林省科技发展计划基金(20200301016RQ)。

摘　　要：本文建立了一种同时检测牛病毒性腹泻病毒(BVDV)和牛冠状病毒(BCoV)2种病原的双重纳米RT-PCR方法。采用BVDV 5′-UTR基因序列(GenBank登录号:AB040132.1)和BCoV的N基因保守序列(GenBank登录号:KX982264.1)设计出一对BVDV特异性引物和一对BCoV的特异性引物,结果表明此方法对牛传染性鼻气管炎病毒(IBRV)、牛呼吸道合胞体病毒(BRSV)、牛副流感病毒3型(BPIV3)和牛轮状病毒(BRV)均无交叉反应,特异性良好。同时,应用此方法检测33份腹泻牛的临床样品和25份腹泻鹿的临床样品,结果得出BVDV、BCoV和2种病原混合感染的牛样品阳性率分别为15.15%、36.36%和6.06%。鹿样品感染BVDV、BCoV和2种病原混合感染的阳性率分别为28%、8%和8%。此外,敏感性试验结果为常规RT-PCR敏感性的10倍,最低检出限为1 fg。研究表明,本文建立的该方法快速、灵敏、简捷,具有较强的特异性、敏感性,可适用于大批量临床样品检测,为反刍动物相关病毒性腹泻的诊断和防控提供一定技术支撑。In this study,a dual nano-RT-PCR method was developed for the simultaneous defection of bovine viral diarrhea virus(BVDV)and bovine coronavirus(BCoV).The 5′-UTR gene sequence of BVDV(GenBank accession No.:AB040132.1)and the conserved N genotype sequence of BCoV(GenBank accession No.:KX982264.1),a pair of specific primers for BVDV and a pair of specific primers for BCoV were designed.The results showed that the method had good specificity but no cross-reaction with bovine infectious rhinotracheitis(IBRV),bovine respiratory syncytial virus(BRSV),bovine parainfluenza type 3(BPIV3)and bovine rotavirus(BRV).At the same time,the method was used to detect 33 clinical samples of diarrhea cattle and 25 clinical samples of diarrhea deer.The results showed that the positive rates of BVDV,BCoV and mixed infection were 15.15%,36.36%and 6.06%,respectively.The positive rates of BVDV,BCoV and mixed infection were 28%,8%and 8%,respectively.In addition,the sensitivity test results were 10 times that of the conventional RT-PCR,and the minimum detection limit was 1 FG.In conclusion,this method was rapid,sensitive,simple,and had strong specificity and sensitivity.It might be applied to the detection of large quantities of clinical samples and provide technical support for the diagnosis,prevention and control of ruminant viral diarrhea.

关 键 词：牛病毒腹泻病毒 牛冠状病毒 双重纳米RT-PCR 

分 类 号：S855.3[农业科学—临床兽医学]