Development of innovative PCR and fluorescence activated cell sorting methodologies for the detection of a range of foodborne pathogens.

The early and rapid detection of the Enterobacteriaceae, Staphylococcus aureus and Listeria monocytogenes is desirable to prevent foodborne outbreaks. In this thesis, novel molecular and flow cytometric based methods were developed for their qualitative and quantitative detection. For S. aureus and L. monocytogenes, two highly specific primer sets were selected for application in simplex and duplex SYBR Green-based real-time PCR assays. Melting curve analysis confirmed the absence of unspecific amplifications and the generation of two PCR products with distinct melting temperatures. The duplex method was successfully applied to spiked food samples after 18 h Buffered Peptone Water enrichment. For the Enterobacteriaceae, a screening real-time PCR protocol using LUX™ primers was developed after primer selection to optimise specificity. This method provided comparable data with a commercial molecular detection system when analysing infant formula milk samples. A novel strategy was developed to improve real-time PCR detection of contaminants based on quantitative standards generated by Fluorescence Activated Cell Sorting (FACS), with S. aureus as the reference microorganism. Optimal PCR efficiency was observed with these standards compared with calibrants prepared with genomic or plasmid DNA. Once storage and temperature conditions are further optimised, FACS generated standards may provide an alternative method to improve accuracy of PCR assays for bacterial quantification. PCR and FACS-based methods were then applied for enumeration of viable foodborne pathogens swabbed from artificially contaminated food-processed surfaces. Sample preparation used Swab Extraction Tube Systems (SETS) for bacterial recovery and Propidium Monoazide (PMA) to remove DNA from dead cells. No significant difference was found between PMA real-time PCR and plate counts. The developed PMA real-time PCR methods may be suitable for samples containing high bacterial loads or for monitoring of disinfection efficacy. Overall, the novel approach of combining PCR and FACS-based methodologies represents valuable progress in the rapid and accurate detection of foodborne pathogens.