Selection, identification and application of a DNA aptamer against Listeria monocytogenes

Abstract

Listeria monocytogenes is a pathogenic bacterium capable of causing severe disease leading to high hospitalization and case fatality rates. Here, we report the use of Systemic Evolution of Ligands by Exponential Enrichment (SELEX) to screen DNA aptamers that recognize L. monocytogenes with high affinity and specificity. Whole L. monocytogenes were incubated with a library of single-stranded DNA (ssDNA) aptamers. The aptamers that bound to L. monocytogenes were isolated using centrifugation, PCR amplified, and purified to yield an enriched ssDNA pool suitable for further rounds of selection. Aptamers with a dissociation constant (Kd) of 48.74 ± 3.11 nM were isolated after eight rounds of selection. An aptamer-based fluorescent bioassay was used to examine the binding ability of the selected aptamers to L. monocytogenes. To demonstrate the potential use of the aptamers in the quantitative determination of L. monocytogenes, a sandwich-type structure fluorescent bioassay with the aptamer A 15 was developed. The assay had a limit of detection (LOD) of 75 cfu/mL and a wide linear range from 102 to 107 with a correlation coefficient of 0.9937. We believe that the design of aptamer-based molecular probes that do not require the labour-intensive steps of isolating and purifying complex markers or targets represents a potentially useful method in the rapid screening and detection of foodborne pathogens.