猪传染性胸膜肺炎快速诊断方法

Establishment of PCR Assay on Diagnosis for Porcine Infectious Pleuropneumonia

针对猪胸膜肺炎放线杆菌的外膜脂蛋白(om lA)基因序列设计了1对特异性引物,研究了猪胸膜肺炎PCR诊断方法;探索了DNA的快速提取方法、PCR反应最适条件、特异性和灵敏度。结果显示:猪胸膜肺炎的6个分离株均能扩增出1个960 bp特异性条带,而对大肠杆菌、金黄色葡萄球菌的扩增结果为阴性;高浓度副猪嗜血杆菌6个分离株的菌液虽然有扩增产物,但是电泳谱带与猪胸膜肺炎明显不同。灵敏度测试表明,APP菌液最低检出浓度为7.7×105个/mL。该方法有望成为应用于猪传染性炎的快速诊断和流行病学调查的新方法。

A pair of primer was designed to detect Actinobacillus pleuropneumoniae （APP） according to a conservative sequence which obtained by comparing six outer membrane lipoprotein （omlA） gene. It was researched that included PCR optimum reaction condition, specificity, sensitivity and DNA purification method rapidly for diagnosis of APP. The results indicated that 960bp fragment was produced by PCR with the primers and DNA from six strains of APP. The specificity research showed that could not amplified DNA fragment by PCR in the samples of E. coli. and Staphylococcus aureaus. And there are fragments produced in Haemophilus parasuis, but the fragments molecular size was different from APP and could be distinguished easily. The DNA extracted by the boiled method can be used to this PCR detection. So it will be more convenient and shorten the times for the diagnosis. The sensitivity in this experiment is 7.7 × 105 APP per mL. The method can be applied to diagnose rapidly for porcine contagious pleuropneumonia and to investigate epidemiology of livestock.