兔源F型多杀性巴氏杆菌荧光定量PCR检测方法的建立

Development of a real-time PCR assay for detection of Pasteurella multocida serogroup F in rabbits

为建立特异性强且敏感性高的兔源F型多杀性巴氏杆菌(Pm)快速检测方法,本实验根据F型Pm fcbD基因的保守序列设计特异性引物和探针,经反应条件优化,建立了检测兔源F型Pm的荧光定量PCR方法。利用该方法检测兔源F型Pm、兔源A和D型Pm、支气管败血波氏杆菌、肺炎克雷伯菌、大肠杆菌、魏氏梭菌和金黄色葡萄球菌等临床常见兔源病原菌,结果显示,该方法仅对兔源F型Pm检测为阳性,其余病原菌均为阴性,特异性强。利用该方法分别检测10倍倍比稀释(1×10^(8)拷贝/μL~1×10^(0)拷贝/μL)的兔源F型Pm基因组DNA,进行敏感性试验,结果显示该方法的检测限为10拷贝/μL,敏感性分别是已报导的双重PCR方法和环介导等温扩增方法的100倍和10倍,敏感性高。利用该方法对不同稀释度的质粒标准品进行批内和批间重复性试验,结果显示,批内和批间变异系数均小于3%,重复性好。利用该方法检测64份已知结果的临床样品,结果显示该方法的检测结果与已报导的双重PCR方法和环介导等温扩增方法检测结果的符合率均为100%,准确性高。本研究首次以fcbD为靶基因建立兔源F型Pm的荧光定量PCR检测方法,为兔源F型Pm的检测提供了有力的技术手段。

To develop a specific and sensitive method for rapid detection of Pasteurella multocida(Pm)serogroup F in rabbits,a real-time PCR assay was developed with a set of primers and probe targeting the conserved sequences of fcbD gene of Pm serogroup F.The results showed that the assay was specific for the rabbit originated Pm serogroup F and had no cross-reaction with Pm serogroups A and D,Bordetella bronchiseptica,Klebsiella pneumonia,Escherichia coli,Clostridium welchii or Staphylococcus aureus that were isolated from rabbits.The assay was sensitive,and the detection limit of the assay was 10 copies/μL of rabbit originated Pm serogroup F genomic DNA,which was 100-and 10-fold higher than those of previously reported duplex PCR assay and loop-mediated isothermal amplification assay,respectively.The assay was repeatable,and the coefficients of both intra-and inter-assay variations were less than 3%.Moreover,the assay was accurate,and the results of the assay based on the 64 clinical samples showed 100%consistency with the results of reported duplex PCR assay and loop-mediated isothermal amplification assay.This study for the first time developed a real-time PCR assay based on the fcbD gene,which will provide an efficient method for the rapid detection of Pm serogroup F in rabbits.