In this paper, a high-affinity ssDNA aptamer binding to Salmonella typhimurium was obtained by a whole-bacterium-based Systemic Evolution of Ligands by Exponential Enrichment (SELEX) procedure. After nine rounds of selection with S. typhimurium as the target, a highly enriched oligonucleotide pool was sequenced and then grouped into different families based on primary sequence homology and secondary structure similarity. Eleven sequences from different families were selected for further characterization via flow cytometry analysis. The results showed that the sequence ST2P demonstrates affinity for S. typhimurium much more strongly and specifically than other sequences tested. The estimated Kd value of this particularly promising aptamer was 6.33 ± 0.58 nM. To demonstrate the potential use of the aptamers in the quantitative determination of S. typhimurium, a fluorescent bioassay with the aptamer ST2P was prepared. Under optimal conditions, the correlation between the concentration of S. typhimurium and fluorescent signal was found to be linear within the range of 50-10(6) cfu/mL (R(2) = 0.9957). The limit of detection (LOD) of the developed method was found to be 25 cfu/mL. This work demonstrates that this aptamer could potentially be used to improve the detection of S. typhimurium.

A whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules to identify DNA aptamers demonstrating specific binding to Vibrio parahemolyticus. FAM-labeled aptamer sequences with high binding affinity to V. parahemolyticus were identified by flow cytometric analysis. Aptamer A3P, which showed a particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K d ) of 16.88 ± 1.92 nM. Binding assays to assess the specificity of aptamer A3P showed a high binding affinity (76%) for V. parahemolyticus and a low apparent binding affinity (4%) for other bacteria. Whole-bacterium SELEX is a promising technique for the design of aptamer-based molecular probes for microbial pathogens that does not require the labor-intensive steps of isolating and purifying complex markers or targets.

Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin produced by Fusarium graminearum on maize and barley. Because most current methods of ZEN detection rely on the use of low-stability antibodies or expensive equipment, we sought to develop a rapid, low-cost determination method using aptamers instead of antibodies as the specific recognition ligands. This work describes the isolation and identification of single-stranded DNA (ssDNA) aptamers recognizing ZEN using the modified systematic evolution of ligands by exponential enrichment methodology based on magnetic beads. After 14 rounds of repeated selection, a highly enriched ssDNA library was sequenced and 12 representative sequences were assayed for their affinity and specificity. The best aptamer, 8Z31, with a dissociation constant (K(d)) of 41 ± 5 nM, was successfully applied in the specific detection of ZEN in binding buffer and in real samples based on a magnetic separation/preconcentration procedure. This analytical method provided a linear range from 3.14 × 10(-9) to 3.14 × 10(-5) M for ZEN, and the detection limit was 7.85 × 10(-10) M. The selected aptamers are expected to be used in the potential development of affinity columns, biosensors, or other analytical systems for the determination of ZEN in food and agricultural products.