摘要
目的根据GenBank中编码大肠杆菌16SrRNA的基因、rfbE(O157抗原基因)、vt1(Vero毒素1基因)、vt2(Vero毒素2基因)和eaeA(紧密素基因)5种基因的特异性序列,设计合成5对引物,建立了快速鉴定大肠杆菌O157的多重PCR方法。该方法的检测敏感度为细菌纯培养物为104CFU/ml,含菌3-4CFU/g鸡肉组织样品经预增菌8h后能检出。用此方法检测2000-2004年间从动物组织和粪便中临床分离的64株菌株,结果10株鉴定为大肠杆菌O157,与血清凝集试验结果完全吻合。其中8株能扩增上述5条特异性条带,与预期产物完全一致,2株能扩增出除vt1基因外的4条特异性条带,其它54株菌株只能扩增出大肠杆菌16SrRNA。该方法能直接鉴定产多种毒力因子的大肠杆菌O157,特异性强,为检测和鉴定大肠杆菌O157提供了一个新方法。
A multiplex PCR was developed by using 5 sets of primers that identifies the sequences of E coli 16s rRNA, O157 antigen （rfbE）, Vero toxin 1 （vtl）, Vero toxin 2 （vt2） and intimin（eaeA）. Sensitivity of the assay was 10^4 CFU/ml of bacteria samples and 3-4 CFU/g of chicken meat that underwent enrichment culturing for 8 hours. The method was then used for the examination of 64 E. coli isolates recovered from animal products and faeces. Among them, 10 were rfbE positive. 8 E. coli isolates of the positive strains possessed the five genes, while the other two positive strains possessed four genes except vtl gene. Moreover, 54 E. coli isolates only possessed the sequence of 16S rRNA. This multiplex PCR assay successfully identifed the Vero toxin-producing Escherichia coli （VTEC） and their main virulence genes, and may be applied to rapid and sensitive detection of E. coli O157 in food when combined with an enrichement step.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2007年第4期351-354,共4页
Chinese Journal of Zoonoses
基金
上海市农委重点攻关项目(No.200411-4)
