| Summary: | Conventional microbiological methods are slow, labour intensive and are unable to meet the demands for rapid food testing. Molecular methods, such as PCR, offer a rapid, sensitive and specific means of detecting pathogens, however loss of sensitivity and lack of robustness have been reported when PCR is applied to heterogeneous and complex food matrices. The aim of this study was to establish a rapid, reliable and sensitive molecular method to detect pathogens in food samples. Real-time PCR assays for the detection of Campylobacter jejuni and coli, Clostridium perfringens, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica and Staphylococcus aureus in food enrichment samples were developed. A novel organism was constructed using a gfp gene cloned into the chromosome of a non-pathogenic Escherichia coli. Viable cells of the modified strain were encapsulated in Lenticule discs and used as process control in the PCR assays. MagNA Pure™ automated extraction was shown to be robust and reliable for preparing bacterial DNA from food enrichment broths. The PCR assays and MagNA Pure™ was applied to enrichment broths inoculated with 558 naturally-contaminated food and environmental samples in a field trial. Concordance was found between PCR results and those obtained using standard culture methods. Loss of assay sensitivity or PCR inhibition was detected in 6 % (32) of the enrichment samples. To improve the sensitivity the L monocytogenes hlyA gene PCR was nested. The assay was applied for the sensitive non-cultural diagnosis of listeriosis, with L monocytogenes detected in 15 of 17 clinical samples from patients with suspected listeriosis. In conclusion, these assays provided a high throughput, robust, reliable PCR detection methods that could be used in clinical and food testing laboratories. The methods will be essential in outbreak situations and could be further developed to detecting bacterial pathogens, viruses, parasites, new and emerging pathogens.
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