PCR检测兔支气管败血波氏杆菌方法的建立及应用

Establishment and Application of PCR Assay for the Diagnosis of Bordetella bronchiseptica Infection in Rabbits

为建立准确、快速检测兔支气管败血波氏杆菌(Bordetella bronchiseptica)的PCR方法,根据Gen Bank中波氏杆菌ptxA基因设计1对特异性引物,用建立的PCR方法扩增兔波氏杆菌,可扩增出870 bp的目的片段。该方法特异性好,仅对波氏杆菌有特异性的扩增,而对大肠埃希菌、无乳链球菌、金黄色葡萄球菌、多杀性巴氏杆菌等病原菌的扩增结果呈阴性;敏感性较高,最低检测限可达6.8×10^(-7)μg/m L;用建立的PCR方法检测临床疑似感染支气管败血波氏杆菌病兔的脏器及鼻拭子样品,可扩增出目的条带,且与细菌分离的结果一致。可见,建立的检测方法敏感、特异,可用于临床上兔波氏杆菌病的快速诊断及鉴定。

To establish accurate and rapid PCR method for detecting Bordetella bronchiseptica（ Bb） in rabbits,a pair of specific primers was designed based on ptxA gene of Bb in Gen Bank. This established PCR method can be used to amplify Bb in rabbits,and the target fragment was 870 bp in length. This method was specific in amplification of Bb,while being negative in amplifying such pathogens as Escherichia coli,Streptococcus agalactiae,Staphylococcus aureus,Pasteurella multocida,etc. Meanwhile,the method was highly sensitive,with minimum detection limit being 6. 8 × 10^-7μg/m L. The PCR method was applied in detecting samples of suspected disease rabbits＇ organs and noses. The amplified target fragment was identical with the result of the bacterial isolation method. Therefore,this detection method is both specific and sensitive and can be applied in rapid clinical diagnosis and identification of Bb in rabbits.