Selection, characterization, and application of DNA aptamers for the capture and detection of Salmonella enterica serovars

• Published in: Molecular and cellular probes 2009-02, Vol.23 (1), p.20-28
• Main Authors: Joshi, Raghavendra, Janagama, Harish, Dwivedi, Hari P., Senthil Kumar, T.M.A., Jaykus, Lee-Ann, Schefers, Jeremy, Sreevatsan, Srinand
• Format: Article
• Language: English
• Subjects: Animals; Aptamers; Aptamers, Nucleotide - genetics; Aptamers, Nucleotide - isolation & purification; Base Sequence; Blotting, Western; Chickens; Deoxyribonucleases - metabolism; Detection; DNA Footprinting; Electrophoretic Mobility Shift Assay; Food borne pathogens; Mass Spectrometry; Membrane Proteins - chemistry; Molecular Sequence Data; Pre-analytical processing; Salmonella; Salmonella enterica - classification; Salmonella enterica - genetics; Salmonella enterica - isolation & purification; SELEX Aptamer Technique
• ISSN: 0890-8508; 1096-1194
• DOI: 10.1016/j.mcp.2008.10.006

Sensitive and specific pre-analytical sample processing methods are needed to enhance our ability to detect and quantify food borne pathogens from complex food and environmental samples. In this study, DNA aptamers were selected and evaluated for the capture and detection of Salmonella enterica serovar. Typhimurium. A total of 66 candidate sequences were enriched against S. Typhimurium outer membrane proteins (OMPs) with counter-selection against Escherichia coli OMPs and lipopolysaccharides (LPS). Specificity of the selected aptamers was evaluated by gel-shift analysis against S. Typhimurium OMP. Five Salmonella-specific aptamer candidates were selected for further characterization. A dilution-to-extinction capture protocol using pure cultures of S. Typhimurium further narrowed the field to two candidates (aptamers 33 and 45) which showed low-end detection limits of 10–40 CFU. DNase protection assays applied to these aptamers confirmed sequence-specific binding to S. Typhimurium OMP preparations, while South-Western blot analysis combined with mass spectrometry identified putative membrane proteins as targets for aptamer binding. Aptamer 33 was bound to magnetic beads and used for the capture of S. Typhimurium seeded into whole carcass chicken rinse samples, followed by detection using quantitative real-time RT-PCR. In a pull-down assay format, detection limits were 10 1–10 2 CFU S. Typhimurium/9 mL rinsate, while in a recirculation format, detection limits were 10 2–10 3 CFU/25 mL rinsate. Reproducible detection at