Molecular detection of foodborne pathogens

Salmonella and Campylobacter are recognised as some of the most important foodborne pathogens worldwide. Human infections have wide health and socioeconomic consequences. Lots of effort has been devoted to increase the knowledge on the prevalence, transmission routes and persistence of these pathogens in the food chain, in order to improve intervention strategies and make more effective the control of production lines and single food items.

To serve this purpose, rapid and reliable detection and quantification methods are imperative. The culture-based standard methods currently applied for detection and enumeration of Salmonella and Campylobacter are time-consuming and laborious. They lack specificity and do not enable detection of viable but non-culturable (VBNC) bacteria.

The focus of the present thesis has been development and validation of PCR-based detection methods for Salmonella and Campylobacter. A conventional PCR-based method for detection of Campylobacter in chicken carcass rinse following 20 h of enrichment in Bolton broth was successfully compared to the ISO standard culture-based method (10272) on 68 naturally infected chickens.

The method was subsequently validated on artificially inoculated pig carcass swab and chicken carcass rinse samples in an international collaborative trial including 12 participating laboratories. A real-time PCR-based method was developed from the conventional method, and this compared favourably to the ISO standard culture-based method (10272) on 12 artificially inoculated and 66 naturally infected chicken carcass rinse samples.

The robustness, detection probability, precision, amplification efficiency and linear range of the method were evaluated on two different thermal cyclers, and the method was proven suitable for quantification. Furthermore, a linear relationship (R2 = 0.94) was established between the threshold cycle values obtained in real-time PCR after enrichment and the culture-based enumeration.

This real-time PCR method was validated according to the recommendations of the Nordic Organisation for Validation of Alternative Microbiological Methods (NordVal) in comparative and collaborative trials, and was approved for detection of Campylobacter in chicken neck skin, cloacal swab and boot swab samples.

A comparison study on probe chemistries for real-time PCR was performed on locked nucleic acid (LNA), minor groove binder (MGB), Scorpion and TaqMan probes. The LNA probe was shown to be the most sensitive probe chemistry in the real-time PCR assay for detection of Campylobacter, producing the highest amplification efficiency.

Choice of probe chemistry was found to impact the sensitivity of PCR assays, and should be considered in an optimization strategy. Finally, the real-time PCR method was evaluated for direct quantification of Campylobacter in chicken carcass rinse samples. A propidium monoazide (PMA) sample treatment was incorporated into the method, enabling PCR detection of viable Campylobacter only.

A strong correlation (R2 = 0.84) was obtained between the Campylobacter counts obtained by PMA-PCR and culture, indicating that the method presents as a reliable tool for producing accurate quantitative data on viable Campylobacter. DNA from dead cells was not detected by the proposed method, however, it recognized the infectious potential of the VBNC state, and would thus be able to assess the outcome and impact of increasingly applied post-slaughter reduction strategies.

A real-time PCR-based method for detection of Salmonella was optimized following a diversified approach to enable the shortest time of analysis possible. Positive effects of the optimization strategy were observed from increasing 1) the sampling volume from the pre-enrichment, 2) the paramagnetic particles applied in the DNA extraction procedure, and 3) the amount of DNA template in the PCR.

This method was subsequently validated according to the recommendations of NordVal in comparative and collaborative trials and was approved as an alternative method for detection of Salmonella in chicken neck skin, minced meat and pig carcass swabs. In conclusion, this thesis presents the development and validation of real-time PCR methods for detection of Salmonella and Campylobacter, and for Campylobacter also quantification of viable cells only.

These validated methods can help the dissemination and implementation of PCR-based methods in control laboratories, and thereby contribute to improved food safety.