一种检测大鲵致病性嗜水气单胞菌的四重PCR法  

Detection of Pathogenic Aeromonas hydrophila in Andrias davidianus by Quadruple PCR

作　　者：凌空[1] 丁诗华[1] 金娟[1] 吴兴镇 

机构地区：[1]西南大学动物科技学院水产科学重庆市市级重点实验室,重庆400715

出　　处：《生物技术通报》2014年第9期201-207,共7页Biotechnology Bulletin

基　　金：重庆市科委科技攻关项目(cstc2013yykfA80006);2013重庆市农委特色效益农业技术支撑项目

摘　　要：建立一种快速诊断致病性嗜水气单胞菌的PCR法。根据嗜水气单胞菌的16S rDNA、外膜蛋白、溶血素及丝氨酸蛋白酶基因设计4对引物,经PCR反应条件优化后进行检测。结果表明,仅嗜水气单胞菌呈阳性,其检测敏感性高,可检测100 fg的DNA模板。20株大鲵源嗜水气单胞菌经四重PCR法检测时,有18株呈阳性。其中,毒力基因种类较多的阳性菌株经人工感染试验和胞外产物活性检测时显示出较高的致病性。利用该PCR法进行的其他检测中,还发现33份人工感染样本全部呈阳性,34份疑似样本有21份呈阳性,说明四重PCR法可应用于临床检测。本研究建立的四重PCR法具有高度敏感性和特异性,可用于嗜水气单胞菌快速诊断。In order to develop a rapid PCR method to detect pathogenic Aeromonas hydrophila in Andrias davidianus, four pairs of primers were designed based on the conservative sequences of 16 S rDNA, outer membrane protein（omp）, hemolysin（hly）and serine protease（ahp）genes. By optimizing of PCR conditions, a quadruple PCR assay was established. The assay had a highly specific detection on pathogenic strains of Aeromonas hydrophila without other irrelative bacteria. This assay had a high sensitivity with the detection limit as low as 100 fg DNA template. From quadruple PCR assay, there were 18 positive results in Aeromonas hydrophila（n = 20）. After artificial infection of Andrias davidianus and the extracellular products assay, the positive strains with more species of virulence genes showed a highly pathogenic activity. This method also was applied in other artificial samples（n = 33）isolated from Andrias davidianus and suspicious samples（n = 34）, resulting in 100% and 21/34 positive results, respectively. In conclusion, the quadruple PCR assay, which has a highly specific and sensitive characteristic, could be applied in rapid diagnosis for Aeromonas hydrophila in Andrias davidianus.

关 键 词：大鲵 嗜水气单胞菌 毒力因子 PCR 

分 类 号：S941.4[农业科学—水产养殖]