猪丹毒杆菌和溶血性曼氏杆菌二重TaqMan荧光定量PCR检测方法的建立

Development of multiplex TaqMan real-time PCR for detection of the Erysipelothrix rhusiopathiae and Mannheimia haemolytica

为建立一种快速、准确检测猪丹毒杆菌和溶血性曼氏杆菌二重TaqMan荧光定量PCR方法,本研究根据猪丹毒杆菌和溶血性曼氏杆菌16S rRNA基因保守序列设计种特异性引物及探针,并优化反应条件,建立了检测猪丹毒杆菌和溶血性曼氏杆菌的二重TaqMan荧光定量PCR检测方法。结果表明,所建方法特异性良好,与其他25种常见细菌均无交叉反应。猪丹毒杆菌检测在2.07×108~2.07×103copies/L范围内具有良好的线性关系,最低可以检测到2.07×102copies/L的标准品阳性质粒,批内和批间变异系数均小于3%;溶血性曼氏杆菌检测在1.07×108~1.07×103copies/L范围内具有良好的线性关系,最低可以检测到1.07×102copies/L的标准品阳性质粒,批内和批间变异系数均小于3%。应用建立的二重荧光定量方法和普通荧光定量方法对47份临床样品进行检测,符合率为100%。本研究结果表明,所建立的方法可用于猪丹毒杆菌和溶血性曼氏杆菌感染的高通量快速诊断以及猪丹毒杆菌和溶血性曼氏杆菌的快速鉴定及定量分析。

To establish a rapid and accurate method for Erysipelothrix rhusiopathiae and Mannheimia haemolytica,a multiplex TaqMan real-time PCR assay was developed for the bacteria detection with two pairs of species-specific primers and two TaqMan probes designed according to the conserved region of the 16 S r RNA gene of the two bacterial species in this study.Under the optimized reaction conditions,the results showed that this method was specific for detecting Erysipelothrix rhusiopathiae and Mannheimia haemolytica and had no cross-amplifications for other pig bacteria.In E.rhusiopathiae detection,the detection limit was 2.07×101copies/L p MD-Er-16 S recombinant plasmids,and the inter-and intra-variation was less than 3%.In M.haemolytica detection,the detection limit was1.07×102copies/L p MD-MH-16 S recombinant plasmids,and the inter-and intra-variation was less than3%.A total of 47 clinical samples were tested by the multiplex real-time PCR assay comparing with conventional real-time PCR,and the coincidence rate were 100%.The results indicated that the detection method could be applied for rapid and high through-put diagnosis of E.rhusiopathiae and M.haemolytica infectionsinpig,andfastidentificationandquantitativeanalysisof E.rhusiopathiaeand M.haemolytica.