生物素标记LT基因探针检测产肠毒素性大肠杆菌

Detection of Enterotoxigenic Escherichia coli with Biotinylated LT Gene Probe

用碱变性法提取重组质粒pEWD299,经Hind Ⅲ酶切后,进行琼脂糖凝胶电泳,用电洗脱法回收LT基因850bp的核酸片段,再用二步法缺口翻译反应制备生物素标记的LT基因探针.通过菌落杂交试验表明,在严格去蛋白和RNA的条件下,该探针与试验中所有产LT和ST,或仅产LT的ETEC发生杂交反应;而与所有仅产ST的ETEC、侵袭性大肠杆菌(EIEC)、普通大肠杆菌、沙门氏菌、志贺氏菌、小肠结肠耶尔森氏菌、霍乱弧菌均无杂交反应.其检测提纯的LT—DNA的敏感性水平为25pg,检测食品模拟标本中产LT大肠杆菌的敏感性达到130个细菌/g的水平.

Plasmid pEWD299 was obtained by the alkaline lysis method. A 850-bp Hind Ⅲ-generated fragment of pEWD299 encoding the B subunit of LT was isolated with argarose gel electroelution. The isolated DNA fragment used as probe was labelled with Bio-11-dUTP by two-steps nick translation. The Bio-DNA probe was highly specific for Escherichia coli (ETEC) when reacted with protein- and RNA-free DNA in a colony hybridization assay. ETEC that produced both LT and ST, and ETEC that produced LT only were positive with Bio-DNA probe. Strains of ETEC that produced ST only, enteroinvasive E. coli (EIEC), E. coll that were non-enterotoxi-genic, Salmonellac, Shigellae, Yersinia enterocolitica, and Vibro cholera were not hybridized with the probe. Two strains of pathogenic E. coli isolated from children with diarrhea were homogeneous in colony hybridization test. As little as 2. 5 pg of target DNA was detected by dot blot hy-brization. ETEC in foods was successfully detected by colony hybridization with the Bio-DNA probe. Sensitivity level was 130 cells per gram.