Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University, Yongin, Republic of Korea.
Biochem Biophys Res Commun. 2012 Jan 6;417(1):414-20. doi: 10.1016/j.bbrc.2011.11.130. Epub 2011 Dec 7.
Escherichia coli (E. coli) O157:H7 is a major foodborne pathogen that causes life-threatening symptoms in humans worldwide. To rapidly and properly identify the pathogen and avoid its toxic effects, ligands which can directly and specifically bind to the virulent E. coli O157:H7 serotype should be identified. In this study, a RNA aptamer-based ligand which can specifically distinguish the pathogen E. coli O157:H7 from others was developed by a subtractive cell-SELEX method. To this end, an RNA library was first incubated with the E. coli K12 strain, and the RNAs binding to the strain were discarded. The precluded RNAs were then used for the selection of O157:H7-specific aptamers. After 6 rounds of the subtractive cell-SELEX process, the selected aptamer was found to specifically bind to the O157:H7 serotype, but not to the K12 strain. This was evidenced by aptamer-immobilized ELISA, real-time PCR analysis, or an aptamer-linked precipitation experiment. Importantly, the isolated RNA aptamer that distinguishes between the virulent serotype and the nonpathogenic strain specifically bound to an O157:H7-specific lipopolysaccharide which includes the O antigen. This novel O157:H7-specific aptamer could be of potential application as a diagnostic ligand against the pathogen-related food borne illness.
产志贺毒素大肠埃希氏菌（Escherichia coli，E. coli）O157:H7 是一种主要的食源性致病菌，可在全球范围内导致对人类生命有威胁的症状。为了快速而正确地识别病原体并避免其毒性作用，应鉴定可直接且特异性结合致病毒力大肠埃希氏菌 O157:H7 血清型的配体。在这项研究中，通过消减细胞 SELEX 方法开发了一种基于 RNA 适体的配体，可特异性区分病原体大肠埃希氏菌 O157:H7 与其他菌株。为此，首先将 RNA 文库与大肠埃希氏菌 K12 株孵育，并去除与该菌株结合的 RNA。然后，将排除的 RNA 用于选择 O157:H7 特异性适体。经过 6 轮消减细胞 SELEX 过程，发现所选适体特异性结合 O157:H7 血清型，但不结合 K12 株。这通过适体固定化 ELISA、实时 PCR 分析或适体连接沉淀实验得到证实。重要的是，区分毒力血清型和非致病性菌株的分离 RNA 适体特异性结合包括 O 抗原在内的 O157:H7 特异性脂多糖。这种新型的 O157:H7 特异性适体可能作为针对与病原体相关的食源性疾病的诊断配体具有潜在应用价值。
