Development of PMAxx-droplet digital PCR method for the absolute quantification of viable Shigella flexneri and Shigella sonnei strains in water

Introduction: . are waterborne pathogens responsible for global diarrheal disease outbreaks via contaminated groundwater, drinking water, and recreational water systems. Their extremely low infectious dose necessitates the development of highly sensitive detection methods capable of distinguishing viable pathogens. Methods: In this study, we developed and optimized a viability-discriminative droplet digital PCR (ddPCR) assay incorporating propidium monoazide (PMAxx) treatment and duplex amplification targeting both chromosomal and virulence plasmid genes. Key reaction parameters-including annealing temperature, primer and probe concentrations, and PMAxx treatment conditions-were systematically optimized. Singleplex and duplex assays were compared to verify amplification consistency. Additionally, three DNA concentration methods (direct centrifugation, PEG precipitation, and a commercial kit) were evaluated for their suitability in field applications using fecal-spiked water samples. Results: with excellent specificity. Duplex amplification showed amplification efficiencies comparable to those of singleplex assays across all targets. The method achieved a detection limit of ≤10 CFU/reaction in fecal-spiked water, and PMAxx treatment effectively suppressed signals from dead cells. Comparative evaluation of concentration methods identified effective protocols suitable for field deployment. Conclusion: in water samples, offering a robust tool for advancing water safety monitoring and public health protection.