Selection and Characterization of Aptamers against Salmonella typhimurium Using Whole-Bacterium Systemic Evolution of Ligands by Exponential Enrichment (SELEX)

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 w...

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Bibliographic Details
Published in:Journal of agricultural and food chemistry 2013-04, Vol.61 (13), p.3229-3234
Main Authors: Duan, Nuo, Wu, Shijia, Chen, Xiujuan, Huang, Yukun, Xia, Yu, Ma, Xiaoyuan, Wang, Zhouping
Format: Article
Language:English
Subjects:
Base Sequence
bioassays
Biological and medical sciences
detection limit
DNA Primers
Flow Cytometry
Food industries
Fundamental and applied biological sciences. Psychology
oligonucleotides
Salmonella typhimurium
Salmonella typhimurium - genetics
SELEX Aptamer Technique - methods
sequence homology
single-stranded DNA
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Summary: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 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 (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.
ISSN:0021-8561
1520-5118
DOI:10.1021/jf400767d