绵羊肺炎支原体和多杀性巴氏杆菌双重PCR检测方法的建立及应用

Development of a duplex PCR assay for double detection of Mycoplasma ovipneumoniae and Pasteurella multocida

根据绵羊肺炎支原体hsp70基因和多杀性巴氏杆菌sp6基因分别设计引物,建立了绵羊肺炎支原体和多杀性巴氏杆菌的双重PCR检测方法,优化条件后进行特异性和敏感性评价,并对160份临床样本进行了检测。结果显示,绵羊肺炎支原体和多杀性巴氏杆菌引物的最佳终浓度分别为30pmol/L和10pmol/L。该方法的特异性较好,对其他无关病原不检出。对绵羊肺炎支原体和多杀性巴氏杆菌的检测下限分别为4.4pg和22pg,与独立PCR的敏感性相同。临床样本的检测结果显示,该方法对绵羊肺炎支原体和多杀性巴氏杆菌的检出率分别为50.6%和47.5%。结果表明,本研究建立的双重PCR方法具有特异性好、敏感性高等特点,为临床上绵羊肺炎支原体和多杀性巴氏杆菌混合感染的快速检测、鉴定以及流行病学调查提供了有用的工具。

The purpose of this study was to establish a duplex PCR method for detection of both Mycoplasma ovipneumoniae and Pasteurella multocida,in order to provide a rapid, accurate and convenient detection tool. A set of primers were designed based on M. ovipneumoniae hsp70 gene and P. multocida sp6 gene. Following optimization of PCR components and reaction conditions, the specificity and sensitivity of the assay were evaluated. Subsequently,a total of 160 clinical samples were tested by the duplex PCR. In result,optimal final concentrations of the primers was 30 pmol/L for M. ovipneumoniae and 10 pmol/L for P. multocida,respectively. The assay was highly specific for M. ovipneumoniae and other pathogens were not detected. The detection limits of the assay were determined to be 4.4 pg for M. ovipneumoniae and 22 pg for P. multocida,respectively,which are as sensitive as individual PCR. Detection of clinical samples by using the duplex PCR demonstrated a 50.6% detection rate for M. ovipneumoniae and 47.5% detection rate for P. multocida. These results suggest that the developed duplex PCR assay is specific and sensitive, and will be useful for clinical detection, identification and epidemiological investigation of M. ovipneumoniae and P. multocida.