Applied Microbiology and Biotechnology

, Volume 97, Issue 8, pp 3677–3686

Selection of DNA aptamers for capture and detection of Salmonella Typhimurium using a whole-cell SELEX approach in conjunction with cell sorting

  • Hari P. Dwivedi
  • R. Derike Smiley
  • Lee-Ann Jaykus
Methods and protocols

DOI: 10.1007/s00253-013-4766-4

Cite this article as:
Dwivedi, H.P., Smiley, R.D. & Jaykus, L. Appl Microbiol Biotechnol (2013) 97: 3677. doi:10.1007/s00253-013-4766-4

Abstract

Alternative ligands such as nucleic acid aptamers can be used for pathogen capture and detection and offer advantages over antibodies, including reduced cost, ease of production and modification, and improved stability. DNA aptamers demonstrating binding specificity to Salmonella enterica serovar Typhimurium were identified by whole-cell-systematic evolution of ligands by exponential enrichment (SELEX) beginning with a combinatorial library of biotin-labeled single stranded DNA molecules. Aptamer specificity was achieved using whole-cell counter-SELEX against select non-Salmonella genera. Aptamers binding to Salmonella were sorted, cloned, sequenced, and characterized for binding efficiency. Out of 18 candidate aptamers screened, aptamer S8-7 showed relatively high binding affinity with an apparent dissociation constant (Kd value) of 1.73 ± 0.54 μM and was selected for further characterization. Binding exclusivity analysis of S8-7 showed low apparent cross-reactivity with other foodborne bacteria including Escherichia coli O157: H7 and Citrobacter braakii and moderate cross-reactivity with Bacillus cereus. Aptamer S8-7 was successfully used as a ligand for magnetic capture of serially diluted Salmonella Typhimurium cells, followed by downstream detection using qPCR. The lower limit of detection of the aptamer magnetic capture-qPCR assay was 102–103 CFU equivalents of Salmonella Typhimurium in a 290-μl sample volume. Mean capture efficiency ranged from 3.6 to 12.6 %. Unique aspects of the study included (a) the use of SELEX targeting whole cells; (b) the application of flow cytometry for aptamer pool selection, thereby favoring purification of ligands with both high binding affinity and targeting abundant cell surface moieties; and (c) the use of pre-labeled primers that circumvented the need for post-selection ligand labeling. Taken together, this study provides proof-of-concept that biotinylated aptamers selected by whole-cell SELEX can be used in a qPCR-based capture-detection platform for Salmonella Typhimurium.

Keywords

SalmonellaDNA aptamersReal-time PCRWhole-cell SELEX

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Hari P. Dwivedi
    • 1
    • 2
    • 4
  • R. Derike Smiley
    • 2
    • 3
  • Lee-Ann Jaykus
    • 1
    • 2
  1. 1.Population Health and Pathobiology, College of Veterinary MedicineNorth Carolina State UniversityRaleighUSA
  2. 2.Department of Food, Bioprocessing and Nutrition SciencesNorth Carolina State UniversityRaleighUSA
  3. 3.U.S. Food & Drug Administration/Office of Regulatory Affairs/Arkansas Regional LaboratoryJeffersonUSA
  4. 4.bioMerieux, Inc.HazelwoodUSA