鼻气管鸟杆菌TaqMan荧光定量PCR检测方法的建立

Development of TaqMan real-time PCR assay for detection of the Ornithobacterium rhinotracheale

为建立鼻气管鸟杆菌(ORT)TaqMan荧光定量PCR检测方法,本研究根据ORT 16S rRNA基因的保守序列设计特异性引物和TaqMan探针,通过优化反应条件、特异性、敏感性、重复性试验和临床样品的检测,建立了ORT TaqMan荧光定量PCR检测方法。实验结果表明:该方法具有良好特异性;最低可以检测到1.09×102拷贝/μL的标准品阳性质粒;批内和批间变异系数均小于3%;23份临床样品检测的阳性率为48%。该方法可以用于鸡ORT感染的早期诊断及ORT的快速鉴定和定量分析。

To establish a rapid and high through-put method for Omithobacterium rhinotracheale detection, a TaqMan real-time PCR assay was developed for the bacteria detection with a pair of primers and one TaqMan probe designed according to the sequence of thel6S rRNA gene of O.rhinotracheale, which was amplified by PCR and cloned into pMD19-T to construct standard recombinant plasmid of pMD-16S. Under the optimized reaction conditions, the test results showed that this method was specific for detecting O.rhinotracheale with a detection limit of 1.09 ×10^2 copies/μL, and had no cross-amplifications for other chicken bacteria, including E.coli, Salmonella, Streptococcus, Staphylococcus, R.anatipestifer, Enterococcus, H.paragallinarum, P.mirabilis and G.anatis. The repeatability tests indicated that the inter-and intra-variation were less than 3%. Therefore, the detection method could be applied in early diagnosis of O.rhinotracheale infections, rapid identification and quantitative assay of the bacteria.