| Background/objective. E. coli O157 is an intestinal pathogen of man that causes diarrhea and the hemolytic-uremic syndrome. It is usually transmitted in food contaminated by bovine feces. Because of its public health importance, novel diagnostic tools with advantages in speed, sensitivity, cost, and field adaptability are in demand. Aptamers, oligonucleotides selected from random pools for desired affinities, have the potential to satisfy these demands. Aptamers can bind targets in a manner similar to antigen-antibody interactions, with similar to superior specificity, dissociation constants, and repertoire. We have performed aptamer selection experiments (SELEX) against E. coli O157 targets with the goal of identifying useful binding species. Methods. In one targeting strategy, E. coli O157 lipopolysaccharide was purified, biotinylated, and immobilized on streptavidin-coated paramagnetic particles. Multiple rounds of SELEX were performed on this target with DNA and RNA aptamer pools. Nitrocellulose immobilization of purified lipopolysaccharide was also attempted for target presentation. In a second targeting strategy, whole E. coli O157 cells were used as a target, and E. coli K-12 and other Enterobacteriaceae were used as negative selection targets. Partitioning of binding ligands was performed by centrifugation. Radiolabeled aptamer binding assays and cloning and sequencing enriched pools were strategies utilized to monitor progress. Results. Selective enrichment of the aptamer pool was not observed during the targeting of immobilized lipopolysaccharide. However, selective enrichment of the pool was observed through 19 rounds of SELEX against E. coli O157 whole cells. Additionally, a binding assay indicated 5:1 preferential binding for O157 over K-12, and the enriched aptamer bound O157 in a significantly greater proportion than the round 0 background. Conclusions. Candidate aptamers for detection of E. coli O157 have been selected. |
