Optimized Viability-ddPCR with Triton X-100 Enhancement for Selective Detection of Live <i>Salmonella enterica</i> and <i>Cronobacter sakazakii</i>

Viability PCR (v-PCR) was optimized using propidium monoazide (PMA) and Triton X-100 for the selective detection of live foodborne pathogens in this study. The colony-forming unit (CFU) method, conventionally used for detecting live foodborne pathogens, is time-consuming. Quantitative PCR (qPCR) and droplet digital PCR (ddPCR) have emerged as rapid alternatives for pathogen detection, which provide accurate detection at low bacterial concentrations. However, these methods cannot distinguish between live and dead bacteria. We investigated the optimization of v-PCR conditions using PMA concentrations (10-200 μM) and Triton X-100 concentrations (0-1%) for <i>Salmonella enterica</i> subsp. <i>enterica</i> and <i>Cronobacter sakazakii</i>. PMA at 100 μM effectively inhibited the amplification of dead bacteria, whereas 1% Triton X-100 enhanced PMA efficiency. The optimized v-ddPCR method successfully distinguished live from dead bacteria, although discrepancies were observed between CFU counts and ddPCR copy numbers. Triton X-100 treatment reduced these discrepancies, bringing the ddPCR copy numbers closer to CFU counts obtained from traditional culture methods. This optimized v-ddPCR protocol provides a rapid and reliable approach for detecting viable foodborne pathogens in food safety applications, that offers enhanced sensitivity compared with conventional culture-based methods.