溶血性曼氏杆菌TaqMan荧光定量PCR检测方法的建立及应用

Development and application of TaqMan real-time PCR assay for the detection of Mannheimia haemolytica

为建立一种快速、准确检测猪溶血性曼氏杆菌TaqMan荧光定量PCR方法,根据溶血性曼氏杆菌16S rRNA基因保守序列设计种特异性引物及探针,优化反应条件,建立了检测溶血性曼氏杆菌的TaqMan荧光定量PCR检测方法。结果表明,以重组质粒pMD-MH-16S为标准品建立的标准曲线在3.06×10^8-3.06×10^1copies/L范围内具有良好的线性关系,最低可以检测到3.06×10^1 copies/L的标准品阳性质粒。该方法与其他24种常见细菌均无交叉反应,批内和批间变异系数均小于3%。应用建立的方法和普通PCR方法对65份临床样品进行检测,阳性率分别为56.92%和21.54%,阳性符合率为100%。本研究结果表明,所建立的方法可用于牛、羊溶血性曼氏杆菌感染的高通量快速诊断以及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 the detection of Mannheimia haemolytica,a TaqMan real-time PCR assay was developed for the bacteria detection with a pair of species-specific primers and one TaqMan probe designed according to the conserved region of 16 S rRNA gene of Mannheimia haemolytica in this study.Under the optimized reaction conditions,the test results showed that this method was specific for detecting Mannheimia haemolytica with a detection limit of 3.06×10^1copies/ L pMD-MH-16 S recombinant plasmid,and had no cross-amplifications for other sheep and goat bacteria.The repeatability tests indicated that the inter-and intra-variation were less than 3%.A total of 65 clinical samples were tested by the real-time PCR assay comparing with the conventional PCR,the positive rates were 56.92% and 21.54%,respectively,and the positive coincidence rate were 100%.The results indicated that the detection method could be applied in rapid and high detection through-put diagnosis of Mannheimia haemolytica infections in cattle,sheep and goat,and fast identification and quantitative analysis of Mannheimia haemolytica.