牛流行热病毒TaqMan实时定量PCR检测方法的建立及应用  

Establishment and application of a TaqMan real-time PCR assay for detection of bovine ephemeral fever virus

作　　者：汪祥斌 贾坤 贾荣荣 李守军 WANG Xiang-bin;JIA Kun;JIA Rong-rong;LI Shou-jun(Guangdong Province Key Laboratory of Veterinary Clinical Integrated Disease Prevention and Control/Guangdong Pet Engineering Technology Research Center,College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]华南农业大学兽医学院,广东省兽医临床重大疾病综合防控重点实验室/广东省宠物工程技术研究中心,广东广州510642

出　　处：《中国兽医学报》2020年第3期491-495,共5页Chinese Journal of Veterinary Science

基　　金：广东省自然科学基金资助项目(2017A030310032);广东省科技计划资助项目(2015B020203005);广东省兽医临床重大疾病综合防控重点实验室项目(2017B030314142);广东省现代农业科技创新联盟建设项目(2018LM2150);广州市珠江科技新星计划资助项目(201610010073)。

摘　　要：通过对牛流行热病毒(bovine ephemeral fever virus,BEFV)中G片段保守区的分析,运用SnapGene设计探针、引物,并优化反应条件。运用最适条件对该方法的敏感性、特异性及重复性进行试验,并检测临床采集的样品。结果显示,本方法特异性较好,其对牛蓝舌病病毒、牛赤羽病病毒进行检测的结果为阴性。该方法线性关系较好,在105~10^10拷贝/μL相关系数达到0.998。该方法较为灵敏,可检测到10拷贝/μL。批内和批间分别重复3次试验得到的变异系数平均值结果较低(<3%)。使用该方法对广东省兽医临床重大疾病综合防控重点实验室收集的109份牛抗凝血样品进行检测,阳性率为7%。本试验成功建立牛流行热病毒TaqMan实时定量PCR检测方法,并将该方法初步运用于牛流行热病毒的临床检测,为BEFV在临床上的快速检测提供更好的方法。To establish a TaqMan real-time PCR method for detection of bovine ephemeral fever virus(BEFV),the primers and probes were designed based on the conserved region of G gene of BEFV by SnapGene,and the PCR reaction conditions were optimized.The specificity test showed that the method for the detection of bovine bluetongue virus and bovine Akabane virus were negative.The method has a good linear relationship in 105-10^10 copies/μL and the correlation coefficient reached 0.998.The method sensitivity was up to 10copies/μL.The results of the reproducibility test showed that the intra-and inter-assay coefficients of variation were both<3%.The TaqMan real-time PCR method was used to detect 109bovine anticoagulant samples which were collected from Guangdong Provincial Key Laboratory of Veterinary Clinical Integrated Disease Prevention and Control,with a positive rate of 7%.The TaqMan real-time PCR method for detection of bovine ephemeral fever virus was successfully established and this method can be used for clinical detection,which laid a good foundation for rapid detection and absolute quantification of BEFV.

关 键 词：牛流行热病毒 TAQMAN探针 实时定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]