非洲猪瘟病毒和胸膜肺炎放线杆菌LyGreen双重荧光PCR检测方法的建立

Establishment of a duplex LyGreen real-time PCR assay for simultaneous detection of African swine fever virus and Actinobacillus pleuropneumoniae

为建立快速、简便同时检测非洲猪瘟病毒(ASFV)和胸膜肺炎放线杆菌(APP)的方法,本研究根据GenBank中公布的ASFV多基因家族(MGF)基因和APP溶血外毒素(apxⅣ)基因序列,分别设计了1对特异性引物,优化反应条件,建立了检测ASFV和APP的LyGreen双重荧光PCR检测方法。结果显示,该方法特异性好,不与其他常见猪病病原体发生交叉反应,检测ASFV和APP的灵敏度分别可达7.3 copies/μL和2.5 copies/μL。本试验建立的LyGreen双重荧光PCR检测方法可对ASFV和APP进行快速诊断,为相关疫病的防控提供了技术支持。

To establish a rapid and convenient method for African swine fever virus(ASFV)and Actiuobacillus pleuropueumouiae(APP)detection,two pairs of specific primers were designed according to the ASFVMGF and APP apxⅣgene sequences collected from GenBank.Reaction conditions were optimized and a duplex LyGreen real-time PCR assay was developed.The results showed that this method exhibited high specificity towards ASFV and APP and did not cross-react with other common cattle disease pathogens.The sensitivity of this method for detecting ASFV and APP reached 7.3 copies/μL and 2.5 copies/μL,respectively.Overall,the duplex LyGreen real-time PCR assay method here was quick and suitable for on-site diagnosis of ASFV and APP,providing a basis for the prevention and control of these two diseases.