Molecular monitoring of disinfection efficacy using propidium monoazide in combination with quantitative PCR

Abstract

One of the major drawbacks of DNA-based microbial diagnostics is its inability to discriminate between live and dead bacteria. Due to the persistence of DNA in the environment after cells have lost their viability, DNA-based assays cannot assess pathogenic risk since signals can originate from both live and dead cells. Presented here is a potential application of the novel chemical propidium monoazide (PMA), which results in the selective suppression of DNA detection from dead cells. PMA can only penetrate dead cells with permeabilized cell membranes. Upon intercalation into the DNA, covalent crosslinkage of PMA to DNA is achieved through light exposure. This modification prevents the DNA from being amplified by PCR. The method, in combination with quantitative PCR as a diagnostic tool, successfully monitored the disinfection efficacy of hypochlorite, benzalkonium and heat on several model pathogens. Threshold cycle numbers increased with increasing disinfection strength after PMA treatment of samples compared to non-PMA treated samples. With some disinfectant-specific differences, monitoring viability loss with membrane integrity as an indicator seemed to be more conservative than monitoring viability loss with plate counts. Loss of viability after short UV-exposure could not be monitored with PMA as UV light affects viability by inducing DNA damage without directly affecting membrane permeability.