Staphylococcus aureus quantification using PMA-ddPCR assay for evaluating the efficacy of simulated disinfection on-site

Disinfection is necessary to block the transmission route of pathogens. Simulated disinfection effect on site can indicate the efficacy of the disinfection process. Therefore, rapid and accurate pathogen quantification after disinfection provides a scientific basis for evaluating its effect. The traditional plate count method used to evaluate the disinfection effect is time-consuming. Here, we established a droplet digital polymerase chain reaction (ddPCR) assay for identifying and quantifying of Staphylococcus aureus by targeting nuc . Our ddPCR method exhibited high specificity and sensitivity, with a limit of detection (LOD) of 36 CFU/µL. Furthermore, propidium monoazide (PMA) was incorparated with ddPCR to differentiate viable and dead cells. Laboratory tests for the identification of a neutralizer of chlorine disinfectants and quantitative germicidal tests of the effect of chlorine disinfectants on a bacterial carrier confirmed that PMA-ddPCR could be used to evaluate the disinfection effect instead of the plate count method. The on-site simulated test revealed that the application of a chlorine disinfection at a concentration of > 500 µg/mL for 30 min was sufficient for disinfecting. Thus, PMA-ddPCR is an acceptable alternative to quantify S. aureus isolates and evaluate the effect of disinfectants. This techniaue is a potentially powerful tool for evaluating the on-site simulate disinfection effect of strong effect disinfectants.