Data supporting the research of Propidium monoazide (PMA) pre-treatment enables qPCR-based pathogen survival monitoring of ozone-treated wastewater effluent

Propidium monoazide quantitative PCR (PMA-qPCR) enables detection of viable bacteria, but its performance in ozonated wastewater requires controlled validation. As membrane filtration isapproved for environmental monitoring, validation of direct PMA application on membrane filters is also needed. Here, we evaluated PMA-qPCR on membrane-filtered samples across five live/dead proportions of four bacterial species, and for E. coli at three initial cell concentrations. PMA-qPCR accurately reflected viable proportions of each species, unlike conventional qPCR. PMA-qPCR accuracy declined at viable cell concentrations below ~104 cells·mL-1. PMA-qPCRwas subsequently applied to ozonated wastewater spiked with E. coli or P. aeruginosa, species with contrasting ozone sensitivity. Spike-ins confirmed accuracy and a lack of PMA toxicity.Ozonation reduced culturable, viable and total E. coli in effluent by 4.4, 2.5, and 1.6 log units, and P. aeruginosa by 1.6, 1.1, and 0.8 log units by culturing, PMA-qPCR, and qPCR, respectively. These results indicate that PMA-qPCR detects viable but non-culturable populations and species-specific survival following ozonation, whereas culturing underestimates, and conventional qPCR overestimates bacterial survival. This study establishes membrane filtration and spike-in quality controls combined with PMA-qPCR as a robust quantitationmethod for viable cells in ozonated effluent, supporting improved microbial risk assessment in disinfected water systems.