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Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX

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Abstract

The need for pre-analytical sample processing prior to the application of rapid molecular-based detection of pathogens in food and environmental samples is well established. Although immunocapture has been applied in this regard, alternative ligands such as nucleic acid aptamers have advantages over antibodies such as low cost, ease of production and modification, and comparable stability. To identify DNA aptamers demonstrating binding specificity to Campylobacter jejuni cells, a whole-cell Systemic Evolution of Ligands by EXponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules. FAM-labeled aptamer sequences with high binding affinity to C. jejuni A9a as determined by flow cytometric analysis were identified. Aptamer ONS-23, which showed particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K d value) of 292.8 ± 53.1 nM with 47.27 ± 5.58% cells fluorescent (bound) in a 1.48-μM aptamer solution. Binding assays to assess the specificity of aptamer ONS-23 showed high binding affinity (25–36%) for all other C. jejuni strains screened (inclusivity) and low apparent binding affinity (1–5%) with non-C. jejuni strains (exclusivity). Whole-cell SELEX is a promising technique to design aptamer-based molecular probes for microbial pathogens without tedious isolation and purification of complex markers or targets.

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Acknowledgments

This work was supported by Food Safety and Research Response Network (FSRRN) funded through United State Department of Agriculture (USDA), Cooperative State Research, Education and Extension Service (CSREES), National Research Initiative, Competitive Grants Program, Epidemiological Approaches to Food Safety, Project grant number 2005-35212-15287. The work represents manuscript FSR10-13 of the Department of Food, Bioprocessing, and Nutrition Sciences, North Carolina State University.The use of trade names in this publication does not imply endorsement by the NorthCarolina Agricultural Research Service or criticism of similar ones not mentioned.

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Author notes

  1. R. Derike Smiley

    Present address: Office of Regulatory Affairs/Arkansas Regional Laboratory, U.S. Food & Drug Administration, 3900 NCTR Road, Building 26, Jefferson, AR, 72079, USA

  2. Hari P. Dwivedi

    Present address: bioMérieux, Inc, 595 Anglum Road, Hazelwood,, MO-63042, USA

Authors and Affiliations

  1. Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, 27606, USA

    Hari P. Dwivedi & Lee-Ann Jaykus

  2. Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, 27695, USA

    Hari P. Dwivedi, R. Derike Smiley & Lee-Ann Jaykus

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  1. Hari P. Dwivedi
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Correspondence to Hari P. Dwivedi.

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Dwivedi, H.P., Smiley, R.D. & Jaykus, LA. Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX. Appl Microbiol Biotechnol 87, 2323–2334 (2010). https://doi.org/10.1007/s00253-010-2728-7

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