猪传染性胸膜肺炎放线杆菌和猪圆环病毒2型二重实时荧光定量PCR检测方法的建立

Development of a Duplex Real-time FQ-PCR for Detection of Actinobacillus pleuropneumoniae and Porcine Circovirus Type 2

以胸膜肺炎放线杆菌(App)apx ⅣA基因和猪圆环病毒2型(PCV-2)ORF2基因为靶基因,分别设计特异性引物和探针,建立App和PCV-2的二重荧光PCR方法。App引物扩增的目的片段大小为132bp,PCV-2为169bp。建立的方法可在同一反应体系中同时对App和PCV-2进行定量检测鉴别,其敏感性分别为1×101拷贝和1×100拷贝,对其他猪病病原核酸的检测为阴性。利用该方法对10份人工感染临床样品进行检测试验,其结果符合率达100%,说明该方法可用于对临床样品中App和PCV-2的检测。

A real-time PCR, based on Taqman hybridization probe technology,was developed and applied to detect Anctinobacillus pleuropneumoniae （App） and Porcine circovirus type 2（PCV-2）. Two pairs of specific primers and two probes labeled were designed for specific amplification of App apxⅣ A and PCV-2 ORF2 genes. The two amplified DNA specific fragments, 132 bp for App and 169 bp for PCV-2 ORF2 genes were cloned into pMD-18T plasmid and transformed into E. coli DH5α. The sensitivity of real time PCR for App and PCV-2 were 1 × 10^1 copies and 1 × 10^0 copies, respectively. The assay was specific for App and PCV-2, and reaction to other organisms was negative. 10 experimental infection clinical samples were subjected to this real-time PCR. The results were matched to the experiment infection. It suggested that this real-time PCR can be used to detect the pathogens of App and PCV-2 in clinical samples.