兔多杀性巴氏杆菌和支气管败血波氏杆菌复合PCR方法的建立及临床应用

Development and clinical application of multiple PCR assay for detection of Pasteurella multocida and Bordetella bronchiseptica infections in rabbits

根据Gen Bank中多杀性巴氏杆菌ATP合成酶基因(atp B)的保守序列设计特异性引物,建立巴氏杆菌PCR检测方法,并与已建立的支气管败血波氏杆菌PCR检测方法进行合并,经反应条件的优化,成功建立多杀性巴氏杆菌、支气管败血波氏杆菌复合PCR诊断方法。该方法检出的最小多杀性巴氏杆菌和支气管败血波氏杆菌菌数分别为40 CFU和4 CFU;对兔源产气荚膜梭菌、大肠杆菌、猪胸膜肺炎放线杆菌、猪鼻支原体、链球菌的检测结果均为阴性,说明该方法具有良好的特异性。用该方法对中国4个省份采集的临床鼻拭子样本共712份进行检测,结果显示,多杀性巴氏杆菌阳性率为25.56%,支气管败血波氏杆菌阳性率为34.55%,双阳性率为13.20%。该复合PCR方法能快速、敏感地从家兔鼻拭子样品中检出多杀性巴氏杆菌和支气管败血波氏杆菌,为临床疾病检测和流行病学调查提供了技术保障。

A pair of specific primers was designed according to the sequences of Pasteurella multocida （19n1） ATP synthase （atpB） in GenBank to establish the P. multocida PCR method. Combined with the PCR method established before for detection of Bordetella bronchiseptica（Bb） infection, a muhiple PCR method was successfully developed through the optimization of reaction conditions. The sensitivities of the multiple PCR method for detecting Pm and Bb were 40 CFU and 4 CFU, respectively. No cross reaction was detected by the multiple PCR for Clostridium perfringens, Escherichia coli , Actinobacillus pleuropneumonia , Mycoplasma hyorhinitis, and Streptococcus suis. In 712 samples of rabbit nasal swab, the positive rates of Pm and Bb in fections were 25.56% and 34. 55%, respectively, and the double positive detection rate of Pm and Bb infections was 13.20%. The nasal swab samples of Pm and Bb infection in rabbits could be detected rapidly and sensitively by the multi- plex PCR method, which provided a technical support for clinical diagnosis and epidemic investigation of Pm and Bb.