| As the causative agent of listeriosis, a severe food-borne disease, Listeria monocytogenes is a major problem for the food industry. The need for pre-analytical sample processing to facilitate concentration of pathogens from complex sample matrices is a well accepted pre-requisite to eventual "real-time" detection. Many ligands (antibodies, peptides, phage binding proteins, nucleic acid aptamers) have been either used or proposed to facilitate this process. Aptamers are small, single-stranded (ss) DNA or RNA molecules that bind with high affinity and specificity to various molecular targets. This dissertation describes the development of (ss) DNA aptamers selected for their binding affinity to Listeria species.;In the first study, SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used to select a biotin-labeled ssDNA aptamer specific for Listeria. After multiple rounds of selection and counter-selection, aptamers with binding affinity to L. monocytogenes were separated, sequenced, and characterized by flow cytometry. 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. The dissociation constant (Kd) of aptamer Lbi-17, selected for its high binding affinity for Listeria spp. and relatively low binding affinity for non-Listeria spp., was 35.7+/-8.0 microM. 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 microl buffer, with a capture efficiency of 26-77%. Increasing assay volume to 10 and 50 ml resulted in reduced capture efficiency and higher limits of detection, to 2.7 and 4.8 log10 CFU L. monocytogenes/sample, respectively, for the AMC-qPCR assay. Based on these preliminary results, the biotinylated ssDNA aptamers developed here appear to be promising ligands for foodborne pathogen concentration prior to detection using molecular methods.;In the second study, carboxyfluorescein (FAM)-labeled aptamers with binding specificity to L. monocytogenes cells at different growth phases were produced by SELEX using cells in log phase (6 h cultures) or stationary phase (12 h cultures). Four aptamers (LM6-2 and LM6-116, selected by targeting log phase cultures; and LM12-6 and LM12-13, selected by targeting stationary phase cultures) were chosen for further characterization based on their relatively high binding affinities for L. monocytogenes (32-55%) as well as other members of the Listeria genus, including L. innocua, L. ivanovii, L. grayi, L. welshimeri, and L. seeligeri. Aptamer binding exclusivity analysis showed low apparent cross-reactivity with other foodborne bacteria, including E. coli O157:H7, Salmonella enterica and Brochothrix thermosphacta, for which binding efficiencies never exceeded 13%. Minimal cross-reactivity was observed for Bacillus cereus (13-18%). Using a sequential binding assay combined with flow cytometry, it was determined that three of the aptamers bound to one apparent cell surface moiety, while a fourth aptamer (LM6-116) appeared to have a different binding site. Dissociation constants (Kd values) of 106.4 +/- 43.91 nM and 74.4 +/- 52.69 nM were determined for aptamers LM12-6 and LM6-116, respectively. An antibody-based magnetic capture coupled with an aptamer detector (two site binding sandwich assay) was developed as a proof-of-concept assay design. When coupled with qPCR detection of the detector aptamer, the lower limit of detection for the assay was 0.4 log10 CFU L. monocytogenes in 500 microl buffer. This is juxtaposed to a detection limit of 2.4 log10 CFU in 500 microl for immunomagnetic separation coupled with qPCR detection of the hlyQ gene target of L. monoctogenes. With further work, the aptamers reported here may be used in assays designed to capture and detect Listeria spp. from complex sample matrices such as foods or environmental samples. |
