Selection, characterization, and application of DNA aptamers for the capture and detection of Salmonella enterica serovars.
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 10-40CFU. 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/9mL rinsate, while in a recirculation format, detection limits were 10(2)-10(3)CFU/25mL rinsate. Reproducible detection at <10(1)S. typhimurium CFU/g was also achieved in spike-and-recovery experiments using bovine feces. The pull-down analysis using aptamer 33 was validated on 3 naturally infected chicken litter samples confirming their applicability in the field. This study demonstrates the applicability of Salmonella specific aptamers for pre-analytical sample processing as applied to food and environmental sample matrices.
Joshi
Raghavendra
R
Veterinary Population Medicine Department, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108, United States.
Janagama
Harish
H
Dwivedi
Hari P
HP
Senthil Kumar
T M A
TM
Jaykus
Lee-Ann
LA
Schefers
Jeremy
J
Sreevatsan
Srinand
S
D000818:Animals; D052157:Aptamers, Nucleotide / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D015153:Blotting, Western; D002645:Chickens; D018983:DNA Footprinting; D003851:Deoxyribonucleases / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D052156:SELEX Aptamer Technique; D019779:Salmonella enterica / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*
D052157:Aptamers, Nucleotide; D008565:Membrane Proteins; D003851:Deoxyribonucleases