副猪嗜血杆菌和猪传染性胸膜肺炎放线杆菌双重PCR检测方法的建立及应用

Establishment and Application of Duplex PCR for Detection of Haemophilus parasuis and Actinobacillus pleuropneumoniae

针对副猪嗜血杆菌(Hps)16SrRNA序列和猪传染性胸膜放线杆菌(App)ApxIVA基因序列各设计一对引物,分别能扩增大小为822bp和346bp的目的基因片段,建立快速鉴定Hps和App的双重PCR方法。特异性试验表明,该方法对波氏杆菌、巴氏杆菌、大肠埃希菌、沙门菌、金黄色葡萄球菌、猪圆环病毒2型、猪流感病毒、猪繁殖与呼吸综合征病毒的扩增结果均为阴性。敏感性试验表明,该方法与单项PCR的敏感性一致,最低可检测核酸浓度Hps 38pg/mL,App 3.8pg/mL;最低检测细菌含量Hps 8.9cfu,App 26.9cfu。利用该方法检测Hps和App参考菌株、临床分离株、36份临床肺组织样本和人工感染的猪肺组织样本。结果显示,该方法对参考菌株和临床分离株全部检出,36份临床样本中检出Hps 18份,App为阴性,与单项PCR检测结果一致,人工感染组织样本也均能检出。表明该方法适用于Hps和App的临床快速检测。

The duplex PCR was developed for detecting Haemophilus parasuis（Hps）and Actinobacillus pleuropneumoniae（App）.The 821bp and 346bp DNA fragments were specifically amplified,respectively,which were based on 16 S rRNA of Hps gene and App ApxIVA.The assay could not amplify any genes of Bordetella bronchiseptica,Pasteurella multocida,Escherichia coli,Salmonella,Staphylococcus aureus,PCV-2,SIV and PRRSV.The duplex PCR could detect 38pg/mL of Hps,3.8pg/mL of App for DNA,as well as 8.9cfu of Hps and 26.9cfu of App for bacteria.The duplex PCR and bacterial culture were used to analyze lung samples from slaughter house pigs.The duplex PCR was positive in 18 of 36 clinical samples and Hps was isolated from 13 of 36 of these samples,which were consistent with the single PCR method to detect Hps and App.Furthermore,all of reference and clinical strains and artificial samples could be identified by this method.Therefore,the results showed that the duplex PCR is suitable for the detection of Hps and App in clinic.