In vitro selection of Escherichia coli O157:H7-specific RNA aptamer

• Published in: Biochemical and biophysical research communications 2012-01, Vol.417 (1), p.414-420
• Main Authors: Lee, Young Ju, Han, Seung Ryul, Maeng, Jin-Soo, Cho, Yong-Jin, Lee, Seong-Wook
• Format: Article
• Language: English
• Subjects: Aptamers, Nucleotide - chemistry; Aptamers, Nucleotide - isolation & purification; Base Sequence; Escherichia coli; Escherichia coli O157 - classification; Escherichia coli O157 - isolation & purification; Escherichia coli O157:H7; Foodborne pathogen; Ligands; Lipopolysaccharide; Molecular Sequence Data; Nucleic Acid Conformation; O Antigens - isolation & purification; RNA aptamer; SELEX Aptamer Technique; Subtractive cell-SELEX
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2011.11.130

► We perform subtractive cell-SELEX method to identify Escherichia coli O157:H7–RNA aptamer. ► The isolated aptamer specifically binds O157:H7 serotype, but not to the K12 strain. ► O157:H7-specific lipopolysaccharide is the target molecule of the isolated aptamer. 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.