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.
出处
《生物技术通报》
CAS
CSCD
北大核心
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
关键词
大鲵
嗜水气单胞菌
毒力因子
PCR
Andrias davidianus
Aeromonas hydrophila
Virulence factors
PCR
