Purpose: The objective of this study is to use whole-bacterium SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for the selection of aptamers specifically binding to E. coli O157:H7, which could later be used for the development of aptamer-based biosensors for rapid detection of target bacteria.
Methods: The whole-bacterium SELEX procedure included four steps: the binding of aptamer candidates to E. coli O157:H7 cells captured by immunomagnetic beads, the partitioning of bound aptamers from unbound DNA oligonucleotides by magnetic separation, the amplification of bound aptamers with PCR, and finally the regeneration of aptamers as the input for the next cycle. Following all cycles of SELEX, the aptamer pool with the highest binding affinity was cloned and sequenced. The binding affinity of the synthesized aptamer was evaluated by dot ELISA.
Results: A total of 19 cycles of selection and 6 cycles of counter-selection were performed. In counter selection, the aptamers were selected against a mixture of S. aureus, L. monocytogenes and S. Typhimurium. Among twenty colonies that were sequenced, only 3 different sequences were obtained, one of which repeated 16 times and was then synthesized. The results of dot ELISA showed that as the concentration of aptamers increased from 10 nM to 1,000 nM, the binding affinity to E. coli O157:H7 increased.
Significance: Ongoing researches focus on the kinetic analysis of selected aptamer and the comparison between the aptamer and the anti-E. coli antibody in both sensitivity and specificity. The selected aptamer may become a substitute of antibodies in the development of biosensors for rapid detection of E. coli O157:H7.