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Development of ssDNA Aptamers for the Sensitive Detection of Salmonella typhimurium and Salmonella enteritidis

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Abstract

Salmonella enterica subsp. enterica ser. enteritidis and Salmonella enterica subsp. enterica ser. typhimurium are the most common and severe food-borne pathogens responsible for causing salmonellosis in humans and animals. The development of an early and ultra-sensitive detection system is the first critical step in controlling this disease. To accomplish this, we used the cell systematic evolution of ligands by exponential enrichment (Cell-SELEX) technique to identify single-stranded DNA (ssDNA) aptamers to be used as detection probes that can specifically bind to S. enteritidis and S. typhimurium. A total of 12 target-specific ssDNA aptamers were obtained through ten rounds of Cell-SELEX under stringent selection conditions, and negative selection further enhanced the selectivity among these aptamers. Aptamer specificity was investigated using the gram-negative bacteria E. coli and P. aeruginosa and was found to be much higher towards S. enteritidis and S. typhimurium. Importantly, three candidate aptamers demonstrated higher binding affinities and the dissociation constants (Kd) were found to be in the range of nanomolar to submicromolar levels. Furthermore, individual aptamers were conjugated onto polyvalent directed aptamer polymer, which led to 100-fold increase in binding affinity compared to the individual aptamers alone. Taken together, this study reports the identification of higher affinity and specificity ssDNA aptamers (30mer), which may be useful as capture and detection probes in biosensor-based detection systems for salmonellosis.

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Acknowledgments

This work was supported by the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ907052)” funded by the Rural Development Administration and by the “Basic Science Research Program through the National Research Foundation of Korea (NRF)” funded by the Ministry of Education, Science and Technology (2012R1A1A2008516), Republic of Korea.

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Author notes

  1. Hae-Chul Park

    Present address: Veterinary Drug and Biologics Division, Animal and Plant Quarantine Inspection Agency, Gyeonggi-do, 430-757, Republic of Korea

Authors and Affiliations

  1. Department of Chemistry, College of Natural Science, Hanyang University, Seoul, 133-791, Republic of Korea

    Hae-Chul Park, Irshad Ahmed Baig, Sang-Choon Lee, Ji-Young Moon & Moon-Young Yoon

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  1. Hae-Chul Park
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  2. Irshad Ahmed Baig
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Correspondence to Moon-Young Yoon.

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Highlights

Identified ssDNA aptamers that selectively bind to S. enteritidis and S. typhimurium.

The candidate aptamers showed high affinity in the nanomolar to sub micromolar range.

Development of conjugated aptamers (PDAP) showed enhanced affinity and specificity.

The identified short 30-mer DNA aptamers can be useful as sensitive detection probes.

Hae-Chul Park and Irshad Ahmed Baig has contributed equally.

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Figure S1

Scheme of PDAP synthesis. a Schematic representation of PDAP (polyvalent directed aptamer polymer). b Schematic representation of PDAP construction. (1) Aptamers were double modified with 5′-FAM and 3′-SH, where FAM acts as the signal probe and the SH group is used as the anchor molecule between the aptamer and SPDP by disulfide bond formation. SPDP is reactive toward amines through the succinimide group and toward sulfhydryl groups through the pyridylthiol group. (2) SPDP-mediated activation of PDL was done by mixing the SPDP-PDL solution at room temperature. The complex was harvested using a gel filtration column. (3) 5′-FAM and 3′-SH labeled aptamers were added to SPDP activated PDL solution. (DOCX 174 kb)

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Park, HC., Baig, I.A., Lee, SC. et al. Development of ssDNA Aptamers for the Sensitive Detection of Salmonella typhimurium and Salmonella enteritidis . Appl Biochem Biotechnol 174, 793–802 (2014). https://doi.org/10.1007/s12010-014-1103-z

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