Department of Microbiology, Faculty of Science, University of Stellenbosch, Private Bag X1, Stellenbosch, 7602, South Africa.
Faculty of Health and Applied Sciences, Namibia University of Science and Technology, 13 Storch Street, Private Bag, Windhoek, 13388, Namibia.
Appl Microbiol Biotechnol. 2017 Oct;101(19):7371-7383. doi: 10.1007/s00253-017-8471-6. Epub 2017 Sep 5.
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.
吖啶橙（EMA）定量聚合酶链反应（qPCR）、吖啶丙啶（PMA）-qPCR 和结合 qPCR 的 DNA 酶处理被用于确定微生物细胞活力。此外，还分析了不同的 EMA 和 PMA 浓度，以确定哪种染料和浓度能够最佳地识别活细胞。在各自的活力测定中，使用了嗜肺军团菌、铜绿假单胞菌、鼠伤寒沙门氏菌、金黄色葡萄球菌和粪肠球菌的活菌、热处理（70°C 15 分钟）和高压灭菌培养物。对活菌和热处理样品的分析表明，对于 EMA[活菌的对数减少范围为 0.01 至 2.71（热处理）和 0.27 至 2.85（热处理）]和 PMA[活菌的对数减少范围为 0.06 至 1.02（热处理）和 0.62 至 2.46（热处理）]和 DNA 酶处理[活菌的对数减少范围为 0.06 至 0.82（热处理）和 0.70 至 2.91（热处理）]，记录了不同的对数减少，与无活力处理对照相比。根据获得的结果，确定 6 μM EMA 和 50 μM PMA 为最佳染料浓度，因为与无活力处理对照相比，记录的对数减少较低（活菌和热处理样品）。此外，对于 6 μM EMA 浓度记录的结果与 50 μM PMA 和 DNA 酶处理获得的结果相当。因此，EMA-qPCR（6 μM）的使用可能允许快速识别和定量水中的多种完整机会性病原体，这将有益于消毒处理后的常规水质监测。
