[目的]建立兔源支气管败血波氏杆菌(Bb)的快速检测方法，用于兔波氏杆菌病的诊断。[方法]以Bb的fimX基因为靶基因，设计特异性引物并构建质粒标准品。在此基础上建立检测兔源Bb的SYBR Green Ⅰ荧光定量PCR方法，对该方法的特异性、敏感性和重复性进行评价，并将该方法初步应用于临床样品的检测。[结果]荧光定量PCR方法仅对兔源Bb的检测结果呈阳性，对其他常见兔源病原菌的检测结果均呈阴性;对质粒标准品的最低检测限为 13。8 拷贝·μL-1，敏感性是普通PCR方法的 10 倍;批内重复性试验的变异系数小于 2%，批间重复性试验的变异系数小于 3%;对 68 份临床样品进行检测，阳性检出率为 39。71%，比普通PCR方法的高 2。95%。[结论]建立的SYBR Green Ⅰ荧光定量PCR方法特异性强、敏感性高、重复性好，且对临床样品的阳性检出率比普通PCR方法高，为兔源Bb的快速检测和防控提供了技术支持。
Development of a fluorescence quantitative PCR assay for detection of Bordetella bronchiseptica in rabbits
[Objective]The study aimed to develop an assay for the rapid detection of Bordetella bronchiseptica in rabbits.[Method]A SYBR GreenⅠ-based fluorescence quantitative PCR assay was developed by designing primers and constructing plasmid standard based on the fimX gene of B.bronchiseptica.After evaluating its specificity,sensitiveness and repeatability,the assay was prelimina-rily used for the detection of clinical samples.[Result]The assay had no cross-reaction with other rabbit-sourced bacterial patho-gens,and was only positive for B.bronchiseptica in rabbits,with a detection limit of 13.8 copies·μL-1 of standard plasmid,which was 10-fold higher than that of conventional PCR assay.The variation coefficients for intra-test and inter-test were less than 2% and 3%,respectively.The detection rate of 68 clinical samples via the assay was 39.71%,which was 2.95% higher than that of the con-ventional PCR assay.[Conclusion]The fluorescence quantitative PCR assay based on SYBR GreenⅠprovided an alternative meth-od for the rapid detection of B.bronchiseptica in rabbits with high specificity,high sensitivity and good repeatability,which is helpful for the prevention and control of B.bronchiseptica in rabbits.
万方数据
维普
