牛产肠毒素大肠杆菌毒力因子多重PCR检测方法的建立

Identification of virulence factors of enterotoxigenic Escherichia coli strains from calves via multiplex PCR

通过多重PCR扩增产肠毒素大肠杆菌(enterotoxigentic E.coli,ETEC)的毒力因子F41菌毛、K99菌毛和STa肠毒素的编码基因来检测和鉴定ETEC。试验中对影响PCR扩增的dNTP、Mg2+、引物浓度以及退火温度等因素进行优化,在优化条件的基础上,确定多重PCR的特异性和灵敏性,以此建立同时检测ETEC多个毒力因子的多重PCR方法。用该方法对分离于犊牛腹泻和犊牛肠毒血症的7株大肠杆菌进行检测,结果2株为F41、K99和STa阳性,4株为F41、STa阳性,1株为K99、STa阳性。这与玻片凝集试验检测菌毛的结果一致。试验表明,该方法特异性强、敏感性高、简便、快速,适用于临床鉴定和检测牛ETEC菌株。

Enterotoxigenic Escherichia coli（ETEC） with virulence factors of K99 and/or F41 fimbriae and heatstable enterotoxin a（STa） is a common pathogen causing diarrhea and acute enterotoxinemia in calves. Fast and reliable detection of the virulence factors is essential for identification and characterization of ETEC. A multiplex PCR method was developed to identify enterotoxigenic Escherichia coli strains by amplifying genes encoding K99 and F41 fimbriae, heat-stable enterotoxin, The parameters of PCR amplification, including concentration of dNTP, Mg^2＋ ,primers and annealing temperature were optimized. The specification and sensitivity of the multiplex PCR experiment were carried out on the basis of optimized parameters. It suggested that this multiplex PCR method was specific,sensitive and useful tool for identify ETEC strains from calves.