地高辛素标记的DNA探针快速鉴定产不耐热肠毒素大肠杆菌的研究

A Rapid Identification of Heat-Labile Enterotoxigenic Escherichia Colt by Nonradioactive Digoxigenin Labeled DNA Probe

用地高辛素标记的６７０ｂＰ－ＬＴ－ＤＮＡ片段作为探针，用菌落原位杂交法对９株标准参考株进行检测，结果全部相符。对７０株从临床分离的菌株也进行菌落原位杂交．并与３２－Ｐ标记的探针进行比较．结果地高辛素探针的特异性为９７．７％，敏感性为１００％，符合率为９８．６％。从含正常肠道菌的粪便中可以直接检出ＬＴ－ＥＴＥＣ而无须经纯培养。定量试验表明本法最低检出量为６ＣｆｕＬＴ－ＥＴＥＣ／斑点。整个检测过程短．仅须２４～３０ｈ。探针的稳定性好，有效期可长达１年。

A DNA probe based on the sequence of a 760bp internalfragment of CPMM030 plamid was labeled with digoxigeninand used as rapid identification of heat-labile enterotoxigenicEscherichia coli (ETEC-LT ).The probe reacted with LT reference-strains and did not reacted with any LT referencestrains by a colony blot hybridization. 70 strams of bacteriaisolated from clinical patients were detected by colony bolt assay using digoxigenin labeled probe. The sensitivity and specificity of digoxigenin-labeled probe were 100% and 97. 7%. respectively. The crud agreement was98. 6 %. ETEC strainswere readily identified in pure and mixed cultures with nonradioactive hybridization. The detected procedure was complete,withm 24￣30 hours. 6 bacteria could be reacted with digoxigenin-labeled probe. Digoxigenin-labeled probe stored at-20cappeared stable for longer than one year.