Development of Molecular-based Methods to Capture and Detect Salmonella and Campylobacter in Complex Sample Matrices.

摘要

Salmonella and Campylobacter are leading causes of food borne bacterial gastroenteritis. Early detection along the farm- to-fork continuum is critical to the success of food safety measures targeting control of these pathogens. It is well recognized that development of methods to concentrate and purify pathogens from complex sample matrices will enhance the application of real-time detection strategies. This dissertation describes the development of novel surface chemistries for pre-analytical sample processing, along with their application to prepare samples for pathogen detection using quantitative real-time PCR (qPCR). The focus is on the food borne pathogens Salmonella and Campylobacter.;In the first study, a combined immunomagnetic separation (IMS)-qPCR assay for capture and detection of Salmonella was developed. This included design and validation of a homologous internal amplification control (IAC) as a signal for amplification failure. In pure culture experiments, the assay demonstrated log linear amplification between 5.19 to 0.19 log 10 CFU equivalents Salmonella per reaction. Genomic DNA was co-amplified in the presence of ≤7.6 ag of IAC with no impact on detection limits at low target concentrations; IAC amplification was out-competed at higher target concentrations (≤2.19 log10 CFU equivalents). The lower limit of detection of the IMS-qPCR method was 100-10 1 CFU Salmonella per 9 ml artificially contaminated chicken rinsate, with capture efficiency of the IMS step increasing (from 12% to 100%) with decreasing levels of Salmonella (10 5 to 100 CFU/sample). The IMS-qPCR approach offers promise in facilitating detection of Salmonella at levels anticipated in naturally contaminated products.;Alternative ligands such as nucleic acid aptamers offer advantages over antibodies including ease of synthesis and labeling, lower cost of production, and equal or higher target binding affinity. The purpose of the second study was to select fluorescein (FAM) labeled DNA aptamers with selectivity to C. jejuni using a whole-cell SELEX (Systematic Evolution of Ligands by EXponential enrichment) approach. Seven aptamer sequences with binding affinity to C. jejuni A9a were identified and the one (aptamer ONS-23) with highest binding efficiency chosen for characterization. Aptamer ONS-23 displayed a dissociation constant of 292.8 nM with 47.3% of C. jejuni cells (n=200,000) bound using 1.48 microM aptamer solution. Inclusivity/exclusivity studies demonstrated a 25--36% binding efficiency for ONS-23 to multiple C. jejuni strains and low apparent binding (1--5%) with non-C. jejuni strains. The whole-cell SELEX approach was successfully applied and offers the advantage of aptamer selection for microbial cells without prior knowledge of diagnostic markers and the need to purify such markers prior to selection, which may ultimately affect aptamer functionality.;In the third study, biotinylated DNA aptamers with binding affinity to S. Typhimurium were identified using the same whole-cell SELEX method. Two of 18 candidate aptamers showed binding efficiency in the range of 13--14% and one of these (S8-7) was further characterized. Aptamer S8-7 displayed a dissociation constant 1.73 microM with 22% of cells bound at a 6.94 microM aptamer concentration. There was low apparent cross-reactivity of this aptamer with E. coli O157: H7 and Citrobacter brkaaii, but moderate cross-reactivity with Bacillus cereus. In proof-of-concept experiments, the S8-7 aptamer was conjugated to magnetic beads and used for Salmonella capture followed by detection using qPCR. The lower limit of detection of the combined aptamer capture-qPCR assay was 102-10 3 CFU equivalents of S. Typhimurium in 290 microl buffer; capture efficiency ranged from 3--13%. Collectively, this research supports the utility of existing (antibodies) and novel (aptamers) ligands in combination with molecular diagnostics to facilitate the concentration and detection of key food borne pathogens.