Detection of viable and VBNC Salmonella in retail meat using optimized PMAxx real-time PCR

Food contaminated with Salmonella remains a major cause of foodborne illness worldwide, posing a significant public health concern. Rapid detection of viable Salmonella in food is essential for effective food safety management. Viability PCR (vPCR) techniques using photo-reactive dyes such as PMA (Propidium monoazide) or PMAxx have emerged as promising approaches to suppress DNA amplification from membrane-compromised (dead) cells. However, most previous PMA-qPCR studies have mainly evaluated artificially contaminated meat samples, while their performance in naturally contaminated food matrices with complex microbiota remains limited. This study optimized a PMAxx real-time PCR assay for the detection of viable and viable but non-culturable (VBNC) Salmonella and evaluated its application in retail meat samples. The PMAxx real-time PCR technique effectively eliminated signals from 10⁸ CFU/mL of dead cells in culture media samples. The limit of detection (LOD) was 10 CFU/g of viable cells in spiked chicken samples following 18 h of pre-enrichment in Buffered Peptone Water (BPW). Under 0.85% NaCl at 4 °C, Salmonella entered the VBNC state after 32 weeks, while PMAxx real-time PCR still detected signals. The method was evaluated using 33 chicken and pork samples collected from supermarkets and traditional markets. A total of 23 samples tested positive, including 13 samples detected after only 6 h of pre-enrichment. The results show 100% agreement with the traditional culture method performed in accordance with ISO 6579-1:2017. These findings indicate that the PMAxx real-time PCR assay can be applied for the rapid screening of viable Salmonella contamination in fresh food samples.