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目的筛选出能特异性识别大肠埃希菌O157:H7的单链DNA(ss DNA)适配体并进行鉴定。方法利用全菌消减SELEX技术,以完整菌体为靶标菌和消减菌,通过体外筛选方法筛选出大肠埃希菌O157:H7特异性ss DNA适配体。利用荧光分光光度计、激光共聚焦显微镜、克隆测序、Chromas和DNAMAN分析软件,分析适配体的一、二级结构,并对筛选得到的适配体进行特异性鉴定。结果经过7轮全菌消减SELEX筛选,荧光分光光度计检测结果鉴定第六轮文库富集达到饱和;通过测序分析、荧光分光光度计和激光共聚焦显微镜特异性检测,获得1条与大肠埃希菌O157:H7特异性结合的适配体Aptamer1。结论利用全菌消减SELEX技术成功筛选获得大肠埃希菌O157:H7特异性ss DNA适配体。
Objective To screen and identify single-stranded DNA (ss DNA) aptamers that specifically recognize Escherichia coli O157: H7. Methods The whole bacteria was used as target bacteria and abatement bacteria by means of whole bacteria elimination SELEX technique. The Escherichia coli O157: H7 specific ss DNA aptamers were screened by in vitro screening method. The primary and secondary structures of the aptamers were analyzed by fluorescence spectrophotometer, confocal laser scanning microscopy, cloning and sequencing, Chromas and DNAMAN software, and the specific aptamers were identified. Results After seven rounds of SELEX selection, the sixth round of library enrichment was identified by fluorescence spectrophotometer. Satisfactory results were obtained by sequencing analysis, fluorescence spectrophotometer and confocal laser scanning microscopy. Aptamer1, an aptamer1 that specifically binds to bacteria O157: H7. Conclusion The Escherichia coli O157: H7 specific ss DNA aptamer was successfully screened by whole-bacteria reduction SELEX technique.