AWWA WQTC64072

Given the rapid progress in molecular technique development and the discussion about the discrepancy between culturability and viability, there is increasing interest in knowing whether the use of plate counts for water safety monitoring could be replaced or at least supplemented with more rapid real-time molecular assays. The potential techniques include quantitative PCR or DNA-chips for detection and enumeration of specific pathogens or indicator organisms. However, one of the major drawbacks of DNA-based diagnostics is its inability to discriminate between live and dead cells. Due to the persistence of DNA in the environment after cells have lost their viability, DNA-based assays can lead to an overestimation of the pathogenic risk as signals can originate from both live and dead cells. This paper presents the treatment of bacterial samples with the novel chemical, propidium monoazide (PMA), which allows for the selective suppression of the signal from dead cells based on membrane integrity. PMA can only penetrate dead cells with permeabilized cell membranes. Upon intercalation into the DNA, light exposure results in covalent crosslinkage of PMA to the DNA. This modification strongly inhibits PCR amplification and limits molecular diagnostics to those cells with intact membranes which prevented PMA from entering the cytoplasm and consequently do not have PMA bound to their DNA. This method was used to monitor the disinfection efficacy of model pathogens using hypochlorite, benzalkonium chloride and UV. Includes 13 references.