牛支原体、巴氏杆菌A型和化脓隐秘杆菌多重PCR快速检测方法的建立

摘要

本试验旨在建立一种可同时鉴别牛支原体、巴氏杆菌A型和化脓隐秘杆菌的多重PCR方法。分别针对多杀性巴氏杆菌A型特异的hyac-hvaD基因区段、化脓隐秘杆菌的16SrRNA基因上保守区段和牛支原体的UvrC基因设计特异性引物，多重PCR的最佳扩增条件确定为：95℃ 10min预变性；95℃ 1min，56℃ 50s，72℃ 1min，循环30次；72℃ 210min延伸。结果表明，该多重PCR方法可同时扩增出以上三种致病菌的特异性片段，不能扩增出其他病原菌的相关片段；对多杀性巴氏杆菌A型、化脓隐秘杆菌和牛支原体的最低检测浓度分别为8×10^5CFU／mL、8×10^5CFU／mL和4×10^6CFU／mL。同时用该方法检测了牛支原体肺炎患牛的鼻拭子与肺组织，发现12h预增菌后，肺组织检测与牛支原体培养的阳性符合率为92％。对临床样本进行牛支原体分离培养需要3-4d时间，而采用多重PCR方法检测12h预增菌则能在24h内出结果。该多重PCR方法显著加快了临床诊断速度，具有推广应用价值。%A triple-PCR method was developed to detected Mycoplasma bovis, Pasteurella multocida type A, and Arcanobacterium pyogenes simutanously. The primer sets were designed specifically to hyaC--hyaD gene segment ofP. multocida type A, conservative section of 16S rRNA gene ofA. pyogenes and the UvrC gene of M. bovis and screened. Finally, one set of primers for each pathogen was selected, and the condition of the multiple PCR was optimized as follows: 95℃ 10 rain, 95℃ lmin, 56℃ 50s, 72℃ lmin for 30 cycles, 72℃ 10min for extension. The specificity tests showed that this multiple PCR method amplified all three target gene fragments from these three pathogens, and did not yield any products for the DNA templates from other common bacterial pathogens of cattle. Sensitivity tests showed that the sensitivities of this multiple PCR for P, multocida type A and A. pyogenes is 8×10^5CFU/mL, and 4×10^6CFU/mL for M. bovis. The detection of M. bovis in lesioned lung and nasal swabs from diseased beef cattle with M. boris pneumonia with this triple-PCR produced a 92% positive agreement with M. bovis culture for the 12h pre-culture of lung samples. Compared to the time of 3-4 d required by 34.. bovis culture, this multiple PCR combined 12h pre-culture can develop the results within 24h and shows a promising application in clinical diagnosis.