猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌复合PCR检测方法的建立

目的建立可以同时检测猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的快速而可靠的PCR检测方法。方法和结果根据胸膜肺炎放线杆菌的Apx-VIA基因序列、多杀性巴氏杆菌和副猪嗜血杆菌的16SrRNA基因序列设计5条引物。猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌模板的PCR扩增产物大小分别为342bp,485bp和1258bp。复合PCR对1～12型猪胸膜肺炎放线杆菌标准株,6株多杀性巴氏杆菌标准株,1～15型副猪嗜血杆菌以及25株经生化鉴定确认为上述三种细菌的分离株的基因组DNA作为模板进行检测,均获得预期大小的扩增产物。以猪放线杆菌、吲哚放线杆菌等14种常见细菌作为阴性对照进行PCR检测,结果仅有支气管败血波氏杆菌产生了可以和上述三个特异性条带明显区分的PCR产物。复合PCR针对胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的敏感性分别为14pg、34pg和37pg。结论本研究建立的复合PCR特异性好,敏感性高,可以用于猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的快速检测。

Development of a multiplex PCR assay for the identication of Actinobacillus pleuropneumoniae, Pasteurella multocida and Haemophilus parasuis

Objective To develop a rapid and qualified diagnostic test for identification of Actinobacillus pleurop- neumoniae, Pasteurella multocida and Haemophilus parasuis. Methods and Results A multiplex PCR assay was developed using primers derived from Apx-VIA gene sequence of Actinobacillus pleuropneumoniae and 16S rRNA gene sequences of Pasteurella multocida and Haemophilus parasuis . The PCR assay correctly identified all the 12 serovars of Actinobacillus pleuropneumoniae, the 14 serovars of Haemophilus parasuis, 6 Pasteurella multocida stan- dard strains and 25 clinical isolates. 14 other bacteiral species commenly isolated from pigs were also tested. Re- sults showed that only Bordetellar bronchiseptica generated two nonspecific bands that could be easily distinguished from those of Actinobacillus pleuropneumoniae, Pasteurella multocida and Haemophilus parasuis when positive con- trol was added. Sensitivity of the test are 14pg, 34pg and 37pg respectively for Actinobacillus pleuropneumoniae, Pasteurella multocida and Haemophilus parasuis. Conclusions The developed multiple PCR can rapidly identify Actinobacillus pleuropneumoniae, Pasteurella multocida and Haemophilus parasuis, with good specificity and high sensitivity.