Abstract
INTRODUCTION: A quadruplex digital polymerase chain reaction (dPCR) method was developed for the simultaneous detection of Salmonella spp., Shigella spp., Vibrio cholerae, and V. parahaemolyticus in wastewater to enhance pathogen identification velocity and efficiency. This study established detection limits for these bacterial pathogens and validated the method using environmental wastewater samples. METHODS: Specific primers and probes were designed targeting the invA gene of Salmonella, ipaH gene of Shigella, tlh gene of V. parahaemolyticus, and cholera toxin gene ctxA of V. cholerae. The quadruplex dPCR assay underwent rigorous evaluation for analytical sensitivity and specificity. Detection limits were determined using spiked wastewater samples, and the method's effectiveness was assessed through preliminary testing of 60 environmental wastewater samples. RESULTS: The quadruplex dPCR assay was optimized at an annealing temperature of 58°C. In spiked wastewater samples, the detection limits were 390 CFU/100 mL for Salmonella, 11 CFU/100 mL for Shigella, 660 CFU/100 mL for V. cholerae, and 640 CFU/100 mL for V. parahaemolyticus. Analysis of 60 municipal wastewater samples revealed pathogen concentrations ranging from 100.9-14,560 copies/100 mL for Shigella, 86.5-7,329 copies/100 mL for Salmonella, and 84.5-865.7 copies/100 mL for V. parahaemolyticus. CONCLUSIONS: The developed quadruplex dPCR assay demonstrates robust capability for comprehensive surveillance of intestinal bacterial pathogens in wastewater, offering reliable detection even at low concentrations.