Optimization of the viability PCR for accurate detection of Staphylococcus aureus in food samples

Staphylococcus (S.) aureus is a prominent foodborne pathogen that can cause food poisoning due to its staphylococcal toxins. Controlling the viable levels of S. aureus is crucial for ensuring food safety. The detection of S. aureus during routine quality control is still primarily conducted using traditional culture-based methods, which are time-consuming and unable to detect viable but non-culturable cells. Viability PCR (vPCR) - a combination of traditional (or quantitative) PCR with photo-reactive DNA-intercalating dye(s) - has been introduced as an alternative to detect viable cells by excluding those with compromised membranes using molecular methods. Despite the success of the vPCR methodology, avoiding false-positive results remains a significant challenge. To enhance the accuracy of vPCR results for S. aureus, several approaches have been proposed by various researchers in the past decade; however, complete PCR signal suppression of dead cells has not been achieved. In this study, we developed a strategy to detect only viable S. aureus cells by combining double PMA treatment with a low PMA concentration and performing a tube change between the last dark incubation and light exposure to improve the vPCR protocol. For pure cultures, the optimized protocol was able to completely suppress DNA signals from 5.0 × 107 dead cells in a final reaction volume of 200 µl. For artificially contaminated food samples with such a high dead cell count, complete PCR signal reduction was observed in ground pepper, - oregano, and infant milk powder, while ground paprika, - allspice, and - pork exhibited PCR signals close to the detection limit. To simulate conditions in real samples, we artificially contaminated ground paprika, - pork, and milk powder with a low number of viable cells (~1.9 cfu/ml) and a high number of heat-inactivated S. aureus (~4.8 × 10⁶ cells/ml). The results showed that the optimized protocol is effective in detecting only the desired live cells, even in the presence of a high dead cell count. Our findings highlight that vPCR can be an accurate and reliable method with strong potential for high-throughput detection of live S. aureus cells in certain food matrices.