Comparison of EMA-, PMA- and DNase qPCR for the determination of microbial cell viability

Abstract

Ethidium monoazide (EMA) quantitative polymerase chain reaction (qPCR), propidium monoazide (PMA)-qPCR and DNase treatment in combination with qPCR were compared for the determination of microbial cell viability. Additionally, varying EMA and PMA concentrations were analysed to determine which dye and concentration allowed for the optimal identification of viable cells. Viable, heat treated (70 °C for 15 min) and autoclaved cultures of Legionella pneumophila, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus and Enterococcus faecalis were utilised in the respective viability assays. Analysis of the viable and heat-treated samples indicated that variable log reductions were recorded for both EMA [log reductions ranging from 0.01 to 2.71 (viable) and 0.27 to 2.85 (heat treated)], PMA [log reductions ranging from 0.06 to 1.02 (viable) and 0.62 to 2.46 (heat treated)] and DNase treatment [log reductions ranging from 0.06 to 0.82 (viable) and 0.70 to 2.91 (heat treated)], in comparison to the no viability treatment controls. Based on the results obtained, 6 μM EMA and 50 μM PMA were identified as the optimal dye concentrations as low log reductions were recorded (viable and heat-treated samples) in comparison to the no viability treatment control. In addition, the results recorded for the 6 μM EMA concentration were comparable to the results obtained for both the 50 μM PMA and the DNase treatment. The use of EMA-qPCR (6 μM) may therefore allow for the rapid identification and quantification of multiple intact opportunistic pathogens in water sources, which would benefit routine water quality monitoring following disinfection treatment.

Methods to prevent PCR amplification of DNA from non-viable virus were not successful for infectious laryngotracheitis virus

“…This may be due to the high molecular weight of DNAse which may have prevented effective penetration of DNAse into inactivated virus particles with damaged capsid or tegument. In the case of environmental samples, the presence of inhibitors can also hinder the DNAse activity [30]. Addition of proteinase K to the heat treatment did not improve DNAse performance in differentiating viable and nonviable ILTV stocks.…”

Propidium monoazide real-time PCR amplification for viable Salmonella species and Salmonella Heidelberg in pork

“…. PMA could penetrate dead cells and binds covalently to DNA to block PCR amplification(Reyneke et al 2017). In contrast, PMA cannot penetrate viable cells therefore PMA-combined real-time PCR assay could detect viable bacteria on food sample.…”

New insights into the kinetics of bacterial growth and decay in pig manure–wheat straw aerobic composting based on an optimized PMA–qPCR method

“…Propidium monoazide (PMA) combined with qPCR has been proven to be able to characterize X viable (Reyneke et al, 2017). The principle is that PMA as a nucleic acid dye binds the exposed DNA of decayed cells by photoactivation and thereby inhibits qPCRs in decayed bacteria.…”

“…Among the studies of liquid samples (Li et al, 2014;Moreno et al, 2015;Bonetta et al, 2017;Casanovas-Massana et al, 2018;Lu et al, 2018), there is little consensus on optimal PMA concentrations, which range from 5 to 100 lM, or on light exposure time, which ranges from 4 to 60 min. In studies of pure strains (Lee et al, 2015;Yu et al, 2015;Lai et al, 2017;Reyneke et al, 2017), the PMA concentrations ranged widely, from 15 to 100 lM, but the light exposure time used in all the studies was <5 min. In studies of solid samples (Moreno et al, 2015;Cancino-Faure et al, 2016;Youn et al, 2017;Casanovas-Massana et al, 2018), the optimal PMA concentration was at a relatively high level, with a range of 50-100 lM; the maximum PMA concentration was 200 lM; and the light exposure time was relatively long, with a range of 8-20 min.…”