Kimata Keiko, Shima Tomoko, Shimizu Miwako, Tanaka Daisuke, Isobe Junko, Gyobu Yotaku, Watahiki Masanori, Nagai Yoshiyuki
Department of Bacteriology, Toyama Institute of Health, Japan.
Microbiol Immunol. 2005;49(6):485-92. doi: 10.1111/j.1348-0421.2005.tb03752.x.
A one-shot multiplex polymerase chain reaction (PCR) was developed for detecting 12 virulence genes of diarrheagenic Escherichia coli. In order to differentiate between the five categories of diarrheagenic E. coli, we selected the target genes: stx1, stx2, and eaeA for enterohemorrhagic E. coli(EHEC); eaeA, bfpA, and EAF for enteropathogenic E. coli(EPEC); invE for enteroinvasive E. coli(EIEC); elt, estp, and esth for enterotoxigenic E. coli(ETEC); CVD432 and aggR for enteroaggregative E. coli(EAggEC); and astA distributed over the categories of diarrheagenic E. coli. In our multiplex PCR system, all 12 targeted genes (stx1, stx2, eaeA, invE, elt, estp, astA, esth, bfpA, aggR, EAF, and CVD432) were amplified in a single PCR reaction in one tube and detected by electrophoresis. Using our multiplex PCR, the 208 clinically isolated strains of diarrheagenic E. coli in our laboratory were successfully categorized and easily analyzed for the presence of virulence plasmids.
开发了一种单管多重聚合酶链反应（PCR）用于检测致泻性大肠杆菌的12个毒力基因。为了区分致泻性大肠杆菌的五类，我们选择了目标基因：肠出血性大肠杆菌（EHEC）的stx1、stx2和eaeA；肠致病性大肠杆菌（EPEC）的eaeA、bfpA和EAF；肠侵袭性大肠杆菌（EIEC）的invE；产肠毒素大肠杆菌（ETEC）的elt、estp和esth；肠聚集性大肠杆菌（EAggEC）的CVD432和aggR；以及分布在致泻性大肠杆菌各类中的astA。在我们的多重PCR系统中，所有12个目标基因（stx1、stx2、eaeA、invE、elt、estp、astA、esth、bfpA、aggR、EAF和CVD432）在一个管中的单个PCR反应中进行扩增，并通过电泳检测。使用我们的多重PCR，成功对我们实验室中208株临床分离的致泻性大肠杆菌菌株进行了分类，并轻松分析了毒力质粒的存在情况。
