猪胸膜肺炎放线杆菌快速PCR检测方法的建立

Establishment of Quick PCR Test for Detecting Actinobacillus pleuropneumoniae

根据猪胸膜肺炎放线杆菌 (Actinobacillus pleuropneumoniae,APP)的 apx A基因序列设计了 1对可扩增 1个4 2 2 bp片段的特异性引物 ,成功地建立了一种检测 APP的快速 PCR方法 ,并确定了其特异性和灵敏性。对血清 1～ 13型等 13个 APP标准株均能扩增出预期 4 2 2 bp的特异性条带 ;对猪副嗜血杆菌、多杀性巴氏杆菌、支气管败血波氏杆菌、大肠杆菌、葡萄球菌、链球菌的扩增结果为阴性。对 APP菌液最低检出浓度为 6 8CFU/ m L(D6 0 0 为 0 .0 0 3)。 12株临床分离菌的 PCR扩增结果与生化鉴定结果是一致的。该方法能从病料中分离培养 8h的混合菌群中快速检测APP,可用于 APP的快速诊断和流行病学的调查。

A quick polymerase chain reaction test was established successfully.According to the apxⅣA gene sequence,a pair of specific primers which being able to amplificate a 422 bp fragment was designed to detect APP and its accuracy and sensitivity were confirmed.The 422 bp specific band could be amplified with 13 APP standard strains of serotype 1-13 by this PCR test,but could not be amplified with some bacterical strains as Hps,Bordetella bronchiseptica,Bordetella bronchiseptica E.coli,S.aureus,S.suis.68 CFU/mL was the lowest detecting limit for mixed bacterical solution of APP with the D_(600) of 0.003.APP could be detected more quickly and exactly by this PCR test from bacterical admixture having been cultivated for 8 hours.