Selective Detection Of Viable Pathogens In Water Using Propidium Monoazide Combined With Qpcr And Its Application

Fast and accurate detection of pathogenic bacteria in water environment is of great importance for waterborne diseases prevention and water quality safety. Since qPCR is limited by its inability in differentiating viable bacterial cells from nonviable bacterial cells, a novel detection method of quantitative PCR combined with a DNA intercalating dye propidium monoazide (PMA-qPCR) was developed in this research, which could selectively detect viable bacterial cells with intact cell membrane. Then PMA-qPCR was applied to quantification of pathogen indicator bacteria in full-scale wastewater treatment plants (WWTPs), as well as evaluation of disinfection efficacy by chlorine, monochloramine and UV disinfection. The following results were obtained.(1) PMA successfully removed DNA from nonviable bacterial cells in qPCR but posed little effects on qPCR detection of DNA from viable bacterial cells, indicating PMA-qPCR's selectivity in detecting viable bacterial cells. But this selectivity varied for different bacterial strains. (2) In different sample matrix such as influent, secondary effluent and sludge from secondary sedimentation tank in WWTPs, PMA-qPCR performed well in selectively detecting viable bacteria cells, but sludge from primary sedimentation tank significantly inhibited the effectiveness of PMA. (3) No significant difference but a good linear correlation was observed between detection results by PMA-qPCR and culture-based method. The concentrations of E. coli and enterococci in full-scale WWTPs measured by PMA-qPCR were closer to those by traditional culture-based methods than those by conventional qPCR, indicating PMA-qPCR's potential utility and accuracy in practical viable pathogen monitoring in WWTPs. (4) Primary treatment of WWTPs did little to remove both of E. coli and enterococci from wastewater, and secondary treatment successfully reduced these two fecal indicators by around 1.5 log10, revealed by PMA-qPCR, conventional qPCR as well as culture-based method. (5) PMA-qPCR could be used to monitor inactivation efficiency of chlorine and monochloramine on E. coli. The inactivation curves on E. coli obtained by PMA-qPCR were in accordance with the first-order kinetic model with inactivation coefficients of 2.24 L/(mg·min) and 0.0175 L/(mg·min) for chlorine and monochloramine disinfection respectively, both of which were lower than those obtained by culture-based method. It indicates that cell membrane integrity was destroyed more slowly than culturability by these two disinfectants. For UV disinfection, both of PMA-qPCR and conventional qPCR could not response to its inactivation efficiency on E. coli and need further researches.