牛呼吸道疾病多重荧光PCR(探针法)检测方法的建立及应用  

Development and Application of TaqMan Multiplex Fluorescent PCR for Detection of Bovine Respiratory Diseases

作　　者：陶攀 东笑[1] 罗昌俊 张桂红 王贵平[1] 贾爱卿 TAO Pan;DONG Xiao;LUO Changjun;ZHANG Guihong;WANG Guiping;JIA Aiqing(Guangdong Haida Animal Husbandry and Veterinary Research Institute Co.,LTD.,Guangzhou 511400,China;Animal Husbandry and Fisheries Research Center,Guangdong Haida Group Co.,LTD.,Guangzhou 511400,China;College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China;Aba Tibetan and Qiang Autonomous Prefecture Animal Disease Prevention and Control Center,Aba 623099,China)

机构地区：[1]广东海大畜牧兽医研究院有限公司,广州511400 [2]广东海大集团股份有限公司畜牧水产研究中心,广州511400 [3]华南农业大学兽医学院,广州510642 [4]阿坝州藏族羌族自治州动物疫病预防控制中心,阿坝州623099

出　　处：《中国动物传染病学报》2024年第6期114-119,共6页Chinese Journal of Animal Infectious Diseases

基　　金：番禺区产业人才项目(2017-R02-4)。

摘　　要：本实验建立了一种能够同时快速检测牛传染性鼻气管炎病毒、牛病毒性腹泻病毒和牛支原体的三重荧光PCR。分别以牛传染性鼻气管炎病毒gB基因、牛病毒性腹泻病毒的5'-UTR基因和牛支原体的P48基因为靶基因设计了特异性引物和TaqMan探针,建立了多重实时荧光PCR方法,并对该方法的灵敏度、特异性、重复性等进行了验证,同时对临床样本进行了检测。结果表明,该方法灵敏度高能够检测出低至10个拷贝数的样本、特异性强、重复性好,并且成本低,与其它牛病原体无交叉反应,对临床样本的检测优于国家标准方法。该方法适合大量临床样品的检测和疫情监控,为牛呼吸道疾病的病原微生物鉴定和降低养殖经济损失提供科学可靠的方法。Guangdong Haida Animal Husbandry and Veterinary Research Institute Co.,LTD.;Animal Husbandry and Fisheries Research Center,Guangdong Haida Group Co.,LTD.;College of Veterinary Medicine,South China Agricultural University;Aba Tibetan and Qiang Autonomous Prefecture Animal Disease Prevention and Control Center;In this study,a triple fluorescence PCR was developed for the simultaneous detection of Infectious bovine rhinotracheitis virus(IBRV),Bovine viral diarrhea virus(BVDV)and Mycoplasma bovis.Specific primers and TaqMan probes were designed based on gB gene of IBRV,5'-UTR gene of BVDV and P48 gene of M.bovis for development of a multiple real-time PCR method followed by optimization for sensitivity,specificity and repeatability.The results showed that the method had high sensitivity with detection as few as 10 copy numbers,strong specificity without reaction with other bovine pathogens,good repeatability,low cost,and advantage over the national standard method in the testing clinical samples.Therefore,this multiple real-time PCR method developed here was suitable for testing a large number of clinical samples and performing epidemic monitoring,providing a scientific and reliable method for identifying pathogenic microorganisms of bovine respiratory diseases and reducing the economic loss of breeding.

关 键 词：牛传染性鼻气管炎病毒 牛病毒性腹泻病毒 牛支原体 多重荧光PCR 

分 类 号：S858.23[农业科学—临床兽医学]