Institute of Environmental Health, College of Public Health, National Taiwan University, Taipei, Taiwan.
Center for Research on Environmental and Occupational Health, National Taiwan University, Taipei, Taiwan.
J Appl Microbiol. 2010 Aug;109(2):623-634. doi: 10.1111/j.1365-2672.2010.04678.x. Epub 2010 Jan 22.
To optimize ethidium monoazide (EMA) coupled with real-time quantitative PCR (qPCR) and to evaluate its environmental applicability on quantifying viable legionellae in water and biofilm of cooling towers and hot water systems.
EMA (0.9-45.5 microg ml(-1)) and propidium monoazide (PMA, 0.9 and 2.3 microg ml(-1)) combined with qPCR (i.e. EMA-qPCR and PMA-qPCR, respectively) were applied to unheated and heated (70 degrees C for 30 min) Legionella pneumophila to quantify viable cells, which was also simultaneously determined by BacLight Bacterial Viability kit with epifluorogenic microscopic enumeration (BacLight-EM). The effects of nontarget microflora and sample matrix on the performance of EMA-qPCR were also evaluated. In comparison with BacLight-EM results, qPCR with EMA at 2.3 microg ml(-1) was determined as the optimal EMA-qPCR assay, which performed equally well as PMA-qPCR for unheated Leg. pneumophila but better than PMA-qPCR for heated Leg. pneumophila (P < 0.05). Moreover, qPCR with EMA at 2.3 microg ml(-1) accurately quantified viable Leg. pneumophila, Legionella anisa and Legionella-like amoebal pathogens 6 (LLAP 6) without interferences by heated legionellae, unheated nonlegionellae cells and cooling tower water matrix (P > 0.05). As for water and biofilm samples collected from cooling towers and hot water systems, the viable legionellae counts determined by EMA-qPCR were mostly greater than the culturable counts by culture assay but consistently lower than the total cell counts quantified by qPCR.
The qPCR with EMA at 2.3 microg ml(-1) may accurately quantify viable legionellae (including fastidious LLAP 6) and Leg. pneumophila pretreated with superheating and is applicable for water and biofilm samples obtained from cooling towers and hot water systems.
The EMA-qPCR assay may be useful in environmental surveillance for viable legionellae and in evaluation of superheating efficacy against legionellae.
优化吖啶橙单键（EMA）与实时定量 PCR（qPCR）相结合的方法，并评估其在定量水和冷却塔及热水系统生物膜中存活军团菌方面的环境适用性。
将 EMA（0.9-45.5μg/ml）和吖啶橙单键（PMA，0.9 和 2.3μg/ml）与 qPCR（即 EMA-qPCR 和 PMA-qPCR）联用，分别用于未加热和加热（70°C 加热 30 分钟）嗜肺军团菌，以定量活细胞，同时通过 BacLight 细菌活力试剂盒与荧光显微镜计数（BacLight-EM）进行定量。还评估了非目标微生物菌群和样品基质对 EMA-qPCR 性能的影响。与 BacLight-EM 结果相比，确定 EMA 浓度为 2.3μg/ml 时 qPCR 为最佳 EMA-qPCR 检测方法，该方法对未加热的嗜肺军团菌与 PMA-qPCR 性能相当，但对加热的嗜肺军团菌优于 PMA-qPCR（P<0.05）。此外，EMA 浓度为 2.3μg/ml 时的 qPCR 可准确定量无干扰的嗜肺军团菌、嗜肺军团菌类似阿米巴病原体 6（LLAP 6）和加热后的军团菌、未加热的非军团菌细胞以及冷却塔水基质中的活军团菌（P>0.05）。对于从冷却塔和热水系统收集的水和生物膜样品，通过 EMA-qPCR 确定的活军团菌计数大多大于培养法可培养计数，但始终低于 qPCR 定量的总细胞计数。
EMA 浓度为 2.3μg/ml 时的 qPCR 可准确定量经过过热预处理的活军团菌（包括难培养的 LLAP 6）和嗜肺军团菌，适用于从冷却塔和热水系统获得的水和生物膜样品。
EMA-qPCR 检测方法可能对环境中活军团菌的监测以及对军团菌的过热效果评估有用。
