牛多杀性巴氏杆菌和牛支原体双重PCR检测方法的建立及应用

摘要

A dual PCR assay was optimized to simultaneously detect two pathogens of bovine Pasteurellamultocida and Mycoplasma bovis in this article. According to the sequences of Pasteurella multocida kmt1 gene and Mycoplasmabovis oppD/F gene in GenBank. Two pairs of primers were designed to amplify the unique framents.By using two pairs of bacterias speciifc primers, two PCR assay were established to amplify the con-servative regions of the two bacterias, respectively. Consequently, a dual PCR method to detect the two bacterias in one tube was developed. The dual PCR system would amplify a 265 bp fragment for bovine Pasteurella multocida, a 439bp for Mycoplasma bovis simultaneously or separately in the samples, depending on its infection status. But not speciifc band ampliifed from other four rabbit pathogenic bacterias. As little as 1pg of bovine Pasteurellamultocida, and 1pg of Mycoplasma bovis DNA were detected using gel electrophoresis in the dual PCR.To evaluate the dual PCR,78 clinical samples were comparatively detected. The data showed that the dual PCR method being 100%coincidence with the single PCR, and it can be used for this two bacteriases detection and differential diagnosis.%参照GenBank中牛多杀性巴氏杆菌kmt1基因序列（AF016259）和牛支原体oppD/F基因序列（AF130119），设计两对引物，在建立两种细菌单项PCR检测方法的基础上，优化双重PCR反应条件，建立了两种细菌的双重PCR检测方法，用这两对引物对同一样品中的牛多杀性巴氏杆菌、牛支原体核酸为模板进行双重PCR扩增，结果可同时扩增牛多杀性巴氏杆菌和牛支原体的265bp和439bp的特异性片段，而对其他4种牛病原菌的扩增结果均为阴性。敏感性测定结果表明，对牛多杀性巴氏杆菌和牛支原体的最低核酸检出限均为1pg。通过对30份临床病料检测，将建立的双重PCR技术和单项PCR方法进行对比验证，结果显示，两者的总符合率为100%。结果表明，建立的双重PCR检测方法具有特异、快速、准确的特点，可用于对牛多杀性巴氏杆菌和牛支原体的同时检测和鉴别诊断。