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  

Development and Applification of TaqMan Fluorescence Quantitative PCR Assay to Detect Rabbit Bordetella Bronchiseptica

作　　者：钱微[1,2] 刘燕[1] 肖琛闻[1] 韦强[1] 季权安[1] 鲍国连[1] 姚火春[2] 

机构地区：[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

出　　处：《农业生物技术学报》2013年第4期498-504,共7页Journal of Agricultural Biotechnology

基　　金：国家兔产业技术体系岗位专家项目(No.CARS-44-C-2);浙江省重大项目(No.2011C12028);浙江省行业公益项目(No.2011C22013)

摘　　要：为了建立特异、敏感、快速检测兔支气管败血波氏杆菌(Bordetella bronchiseptica,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0.32pg,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。A specific, sensitive and rapid TaqMan fluorescence quantitative PCR was established for testing Rabbit Bordetella bronchiseptica. In present study, a pair of primers and probes were designed from target gene of virulence factors CyaA of Bordetella bronchiseptica. 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 quantitative detection of standards to establish standard curve. The detectable quantity ofBordetella bronchiseptica genomic DNA was 0.32 pg, which was 25 times sensitivity compared with common PCR, there was no cross reaction with common clinical bacteria by TaqMan fluorescence quantitative PCR. The 45 suspected samples were detected by TaqMan fluorescence quantitative PCR or routine PCR. The positive detection rates were 75.5% and 66.7%, respectively, the coincidence rate was 88.2%. The results showed that the TaqMan fluorescent quantitative Rabbit BordeteUa bronchiseptica detection method was specificity, sensitivity and reproducibility. The present study provides an effective sensitive and reproducible method to diagnose and control Rabbit Bordetella bronchiseptica in further research.

关 键 词：兔支气管败血波氏杆菌 CyaA 荧光定量PCR 检测 

分 类 号：S858.28[农业科学—临床兽医学]