兔支气管败血波氏杆菌TaqMan荧光定量PCR检测方法的建立与应用.pdfVIP

Online system: 农 业 生 物 技 术 学 报 Journal of Agricultural Biotechnology 2013, 21(4): 498~504 DOI: 10.3969/j.issn. 1674-7968.2013.04.016 技术改进 Updated Technology 兔支气管败血波氏杆菌TaqMan 荧光定量PCR 检测方法的建立与应用 1，2 1 1 1 1 1* 2 钱微 刘燕 肖琛闻 韦强 季权安 鲍国连 姚火春 1浙江省农业科学院，畜牧兽医研究所，杭州310021 ；2 南京农业大学，动物医学学院，南京310025 *通讯作者，baoguolian@ 163.com 摘 要 为了建立特异、敏感、快速检测兔支气管败血波氏杆菌(Bordetellabronchiseptica, Bb) 的TaqMan 荧光定量PCR 方法，本研究以Bb 的毒力因子CyaA为目的基因设计特异性引物和探针，并将PCR 扩增产 物克隆测序，测序结果与GenBank 上Bb 的CyaA的同源性达100% 。以阳性克隆质粒作为定量检测标准 品建立标准曲线，以提取的Bb 基因组DNA 为模板，进行特异性、灵敏度和重复性实验。该方法对波氏杆 菌基因组DNA 检测最低限为0.32 pg ，灵敏度是普通PCR 的25 倍，与临床常见细菌无交叉反应。对45 份 疑似感染兔波氏杆菌病料的检测表明，TaqMan 荧光定量PCR 和普通PCR 检测阳性率分别为75.6%和 66.7%，两者符合率88.2%。结果表明，建立的TaqMan 荧光定量Bb 检测方法具有较好的特异性、敏感性 和重复性。该方法的建立对Bb 的临床高效诊断，Bb 的防控提供了有效手段。 关键词 兔支气管败血波氏杆菌，CyaA，荧光定量PCR ，检测 Development and Applification of TaqMan Fluorescence Quantitative PCR Assay to Detect Rabbit BordetellaBronchiseptica QIAN Wei1, 2 LIU Yan1 XIAO Chen-Wen1 WEI Qiang1 JI Quan-An1 BAO Guo-Lian1* YAO Huo-Chun2 1 Institute of Animal Husbandry and Veterinarian, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China; 2 College of Verterinary Medicine, Nanjing Agriculture University, Nanjing 210095, China *Corresponding author, baoguolian@ 163.com Abstract A specific, sensitive and rapid TaqMan fluorescence quantitative PCR was established for testing Rabbit Bordetellabronchiseptica. In present study, a pair of primers and probes were designed from target gene of virulence factors CyaAof Bordetellabronchiseptica. Amplified PCR product was cloned and sequenced, the results showed that the homology was 100% compared with the reference sequence published in GenBank. The positive recombinant plasmids were served as quantita