肠致病性大肠杆菌适配体筛选研究

Selection of an aptamer targeted to Enteropathogenic Escherichia coli

中文关键词:  肠致病性大肠杆菌  适配体  Whole bacteria-SELEX技术  荧光分析

英文关键词:Enteropathogenic Escherichia coli  aptamer  whole bacteria-SELEX  fluorescence analysis

中文摘要:

      目的 食源性致病菌引起的食源性疾病已成为全球食品安全面临的巨大威胁, 其中由大肠杆菌引起的食源性疾病最为常见。因此发展快速、准确检测大肠杆菌的方法对于保障食品安全、保护国民健康具有重要意义。方法 应用Whole bacteria-SELEX(指数富集的配体系统进化技术), 以肠致病性大肠杆菌为靶标, 经过15轮的筛选富集, 将所得产物进行克隆测序, 并结合荧光分析考察所得序列的亲和力和特异性, 根据解离常数值分析比较序列的亲和力和特异性, 最终得到2条(Seq.1和Seq.28)与肠致病性大肠杆菌高特异性结合的适配体。结果 适配体Seq.1和Seq.28均对肠致病性大肠杆菌表现出了相对良好的亲和力和特异性(对其他菌的相对亲和力均低于15%), 解离常数分别为45.06±6.797和32.31±6.002 nmol/L。结论 本文利用SELEX技术筛选特异性识别肠致病性大肠杆菌适配体, 具有稳定性高、合成方便、易标记等特点, 可进一步应用于食品中肠致病性大肠杆菌的快速检测。

英文摘要:

      Objective Foodborne diseases caused by foodborne pathogenic bacteria have become a great threat to the global food security. The most common is Enteropathogenic Escherichia.coli (EPEC). Therefore, it will be of great significance to develop rapid and accurate detection method for ensuring food safety and na-tional health. Methods In this work, a whole-bacteria Systemic Evolution of Ligands by Exponential Enrichment (SELEX) method was applied to identify aptamers specific binding to EPEC. After fifteen rounds of selection, a highly enriched oligonucleotide pool was sequenced. The binding affinity and specificity assay were analyzed by fluorescence analysis. Results Two sequences (Seq.1 and Seq.28), which presented high affinity and specificity with the target, were regarded as aptamers of EPEC. The binding affinity of Seq.1 and Seq.28 to the other bacteria was all below 15%. The dissociation constant (Kd value) was 45.06±6.797 and 32.31±6.002 nmol/L, respectively. Conclusion Owing to their high selectivity, stability, and easy synthesis and label, aptamers have application in detecting EPEC in food.