In vitro selection and characterization of ssDNA aptamers by cross-over SELEX and its application for detection of S. Typhimurium.

S. Typhimurium is the most common food-borne pathogen frequently encountered in contaminated food and poses a major threat to public health, animals, and the food industry worldwide. Early and sensitive detection is the prime step to ensuring the microbiological quality of fresh and processed food. We used the cross-over SELEX strategy to select ssDNA aptamers against OM usher protein, FimD that could be employed in the S. Typhimurium detection assay. Altogether, 7 rounds of protein-SELEX, 3 rounds of intermittent counter-SELEX, and 4 rounds of whole bacterial cell-SELEX were undertaken for specific aptamer selection. The aptamer specificity was achieved by performing counter-SELEX with non-target bacterial cells. The enriched pool of aptamers obtained post SELEX process was cloned, sequenced, and further the specificity and affinity were characterized by flow cytometry. The aptamers DFRM-3 and DFRM-10 were specific and showed relatively high binding affinity towards S. Typhimurium with an apparent dissociation constant (Kd value) of 76 nM and 90.01 nM respectively. A dual aptamer-based sandwich assay was developed using biotinylated DFRM-3 aptamer for magnetic capture and AlexaFluor-488 labeled DFRM-10 aptamer for detection of S. Typhimurium. The limit of detection (LOD) of S. Typhimurium by the developed assay was found to be 10 CFU/ml in pure culture and 10 CFU/ml in the artificially contaminated chicken meat samples.