SS-2、APP和PM多重PCR检测方法的建立与应用

Establishment of a multiplex PCR assay for detection of SS-2,APP and PM

猪链球菌2型(SS-2)、猪胸膜肺炎放线杆菌(APP)、多杀性巴氏杆菌(PM)是引起"猪无名高热症"的部分病原菌,为建立可以同时检测SS-2、APP和PM的多重PCR快速诊断方法,根据GenBank中公布的有关基因序列,设计了3对引物分别用于扩增CPS2J(SS-2),ApxIVA(APP),KMT1(PM)基因的片段。敏感性和特异性结果表明,本方法对3种病原菌的最低DNA检测量分别为48.3pg(SS-2),581pg(APP),5pg(PM),而对大肠杆菌、沙门菌、猪附红细胞体、刚地弓形虫的扩增结果均为阴性。35份临床样品的多重PCR检测结果表明,有7例SS-2,5例APP,7例PM,与生化试验的鉴定结果一致。表明所建立的多重PCR方法能够对SS-2、APP和PM单个或混合感染的临床样品进行快速鉴别诊断。

According to the gene sequences in GenBank of Streptococcus suis serotype 2（SS-2）,Actinobacillus pleuropneumoniae （APP） and Pasteruella multocida （PM）,three pairs of specific primers were designed to amplify the three fragments,respectively.A multiplex PCR assay was established to detect SS-2,APP and PM after optimizing the reaction condition.The results of sensitivity and specificity showed that the minimum DNA that multiplex PCR can detect were 48.3 pg （SS-2）,581 pg （APP）,5 pg （PM）.All the predicted fragments were amplified but no band was observed from Escherichia coli,Salmonella choleraesuis,Eperythrozoon suis,Toxoplasma gondii.35 clinical samples were detected by the multiplex PCR.As the results,7 samples were positive for SS-2,5 positive samples for APP,and 7 positive samples for PM.The results showed that the multiplex PCR could be used to simultaneously detect the clinical samples infected with SS-2,APP,PM single-infection or co-infection.