Selection and Characterization of DNA Aptamers with Binding Specificity for Listeria spp. and the Use of DNA Aptamers for Capture of Listeria spp. Prior to the Application of qPCR for Detection

Introduction: More rapid detection of foodborne pathogens could be achieved if the target(s) were concentrated and purified from the sample prior to detection by quantitative PCR (qPCR).  Aptamers [small, single-stranded (ss) DNA or RNA molecules with target binding affinity] are emerging ligands to facilitate pathogen capture.  These demonstrate advantages over traditional capture ligands like antibodies, including reduced cost, ease of production, and high stability.

Purpose: To identify ssDNA aptamers with binding specificity to Listeria monocytogenes and use these for capture and subsequent qPCR detection of the organism. 

Methods: 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 with binding affinity to L. monocytogenes were separated, sequenced, and characterized with the aid of flow cytometry.  One aptamer (Lbi-17) was conjugated to streptavidin-coated magnetic particles and used for capture and subsequent detection of L. monocytogenes using qPCR targeting the hly gene.

Results: Five aptamer candidates were identified, all having binding affinities of 18-24% as evaluated by flow cytometry. Although selected for using L. monocytogenes, these aptamers showed similar binding affinity for other members of the Listeria genus (13-21%), and low binding affinity for non-Listeria species. Aptamer Lbi-17 was chosen for further characterization based on its high binding affinity for Listeria and relatively low binding with non-Listeria spp. The dissociation constant (K_d) of Lbi-17 was 35.7 ± 8.0 µM.  When Lbi-17 was conjugated to magnetic beads and used for capture and detection of L. monocytogenes, capture efficiency (CE) ranged from 15-19%.  A similar CE was observed for IMS-qPCR (13-17%), but the limit of detection for the aptamer-based method was one log lower (better) at 60 CFU/ml.

Significance: This study shows proof-of-concept that ssDNA aptamers can be used for capture and detection of a common foodborne pathogen, with detection limits better than those observed for IMS-qPCR.