Selection and characterization of aptamers against Salmonella Typhimurium using whole-bacterium SELEX
Journal of agricultural and food chemistry | 12 Mar 2013
N Duan, S Wu, X Chen, Y Huang, Y Xia, X Ma and Z Wang
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 K¬d 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 the optimal conditions, the correlation between the concentration of S. typhimurium and fluorescent signal was found to be linear within the range of 50-106 cfu/mL (R2 =0.9957). The limit of detection (LOD) of the developed method were found to be 25 cfu/mL. Our work demonstrates that this aptamer could potentially be used to improve the detection of S. typhimurium.
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