Nucleic acid aptamers for capture and detection of Listeria spp.
The purpose of this study was to identify biotinylated single-stranded (ss) DNA aptamers with binding specificity to Listeria and use these for capture and subsequent qPCR detection of the organism. For aptamer selection, SELEX (systematic evolution of ligands by exponential enrichment) was applied to a biotin-labeled ssDNA combinatorial library. After multiple rounds of selection and counter-selection, aptamers separated, sequenced, and characterized by flow cytometry showed binding affinities to L. monocytogenes of 18-23%. Although selected for using L. monocytogenes, these aptamers showed similar binding affinity for other members of the Listeria genus and low binding affinity for non-Listeria species. One aptamer, Lbi-17, was chosen for development of a prototype capture and detection assay. When Lbi-17 was conjugated to magnetic beads and used in a combined aptamer magnetic capture (AMC)-qPCR assay, the pathogen could be detected at concentrations <60 CFU/500 μl buffer in the presence of a heterogeneous cocktail of non-Listeria bacterial cells, with a capture efficiency of 26-77%. Parallel experiments using immunomagnetic separation (IMS)-qPCR produced the same detection limit but lower capture efficiency (16-21%). Increasing assay volume to 10 and 50 ml resulted in reduced capture efficiency and higher limits of detection, at 2.7 and 4.8 log₁₀ CFU L. monocytogenes per sample, respectively, for the AMC-qPCR assay. Biotinylated ssDNA aptamers are promising ligands for food-borne pathogen concentration prior to detection using molecular methods.
Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA. Electronic address: ssuh@ncsu.edu.
Soo Hwan
SH
Suh
Lee-Ann
LA
Jaykus
D052157:Aptamers, Nucleotide / Q000138:chemical synthesis* / Q000191:economics; D001431:Bacteriological Techniques / Q000379:methods; D001483:Base Sequence; D019921:Biotinylation; D005434:Flow Cytometry; D008089:Listeria monocytogenes / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction
D052157:Aptamers, Nucleotide
Aptamer magnetic capture; DNA aptamer; Immunomagnetic separation; Listeria monocytogenes; Listeria spp; Real-time PCR