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

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• 作者：Nuo Duan ; Shijia Wu ; Xiujuan Chen ; Yukun Huang ; Yu Xia ; Xiaoyuan Ma ; Zhouping Wang
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2013
• 出版时间：April 3, 2013
• 年：2013
• 卷：61
• 期：13
• 页码：3229-3234
• 全文大小：307K
• 年卷期：v.61,no.13(April 3, 2013)
• ISSN：1520-5118

文摘

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 卤 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鈥?0⁶ cfu/mL (R² = 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.

Keywords:

aptamer; Salmonella typhimurium; SELEX; fluorescence