Real-time quantitative PCR combined with propidium monoazide for simultaneous detection and quantification of Escherichia coli, Staphylococcus aureus and Salmonella spp. during processing of animal-based food products

Rapid and sensitive analytical methods are under investigation, both in vitro and in fresh food samples, as alternatives to conventional microbiological culture techniques, aiming to reduce the time required for microbiological quality assessment of food products. Real-time quantitative PCR (qPCR) is a molecular technique extensively researched for rapid pathogen detection in food safety applications. However, the primary limitation of qPCR is its inability to differentiate between living (viable) and dead (nonviable) bacterial cells. To address this limitation, propidium monoazide (PMA) - a viability dye that selectively penetrates damaged cell membranes - was combined with qPCR (PMA-qPCR) to enable exclusive detection of viable cells in food samples. This study aimed to evaluate multiplex qPCR combined with PMA pretreatment for simultaneous quantification of Salmonella spp., Escherichia coli, and Staphylococcus aureus during processing of three food products (Minas Frescal cheese, frozen beef burgers, and frozen oyster fillets), comparing results against traditional culture methods (gold standard), "ready-to-use" microbiological test, and multiplex qPCR without PMA pretreatment. The multiplex PMA-qPCR technique demonstrated a statistically significant agreement with the gold-standard culture methods for quantifying E. coli and S. aureus in the three food matrices tested (Minas Frescal cheese, frozen beef burgers, and raw oyster fillets). However, for Salmonella detection, multiplex PMA-qPCR was more sensitive than microbiological culture methods. Our findings indicate that PMA-qPCR may represent a promising alternative method for enhanced pathogen detection in foods, offering reduced analysis time and decreased non-viable cell amplification, although its effectiveness varies by food matrix.