Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
J Appl Microbiol. 2011 Nov;111(5):1194-204. doi: 10.1111/j.1365-2672.2011.05125.x. Epub 2011 Sep 8.
Ethidium monoazide in combination with quantitative PCR (EMA-qPCR) has been considered as a promising method to enumerate viable cells; however, its efficacy can be significantly affected by disinfection conditions and various environments. In this study, thermal disinfection, osmotic pressure and acids with different pH values were systematically investigated to achieve the optimum conditions.
EMA treatment of pure cultures at low concentration (10 μg ml(-1)) for 20 min resulted in effective differentiation between viable and nonviable bacteria and had no effect on viable cells. Heating at 85°C for 35 min was the optimum condition that yields inactivated Escherichia coli (E. coli) cells that were not detected with EMA-qPCR. Performing EMA treatment in high-salt ion environment (sodium chloride concentration ≥4%) could weaken EMA inhibition effect. Both strong and weak acid solutions could react with EMA, change its absorption spectra and influence EMA inhibition effect. Because of the sublethal acidification injury, underestimation of cell counts were found using EMA-qPCR method, and 40-min incubation in Luria-Bertani medium could completely offset this error.
Our results provided optimum EMA treatment, thermal disinfection and environment conditions for EMA-qPCR and demonstrated the feasibility of this method when enumerating viable cells under varied osmotic pressure and pH environment.
Optimum EMA treatment, thermal disinfection and EMA-treated environment will be successfully applied in EMA-qPCR. Osmotic pressure and acid-induced injury can be detected by EMA-qPCR with optimization.
吖啶橙单碱基嵌入结合定量 PCR（EMA-qPCR）已被认为是一种有前途的方法来对活细胞进行计数；然而，其效果可能会受到消毒条件和各种环境的显著影响。在这项研究中，我们系统地研究了热消毒、渗透压和不同 pH 值的酸，以达到最佳条件。
在低浓度（10 μg ml(-1)) 下对纯培养物进行 20 分钟的 EMA 处理可有效区分活细菌和非活细菌，且对活细胞没有影响。85°C 加热 35 分钟是产生无法用 EMA-qPCR 检测到的失活大肠杆菌（E. coli）细胞的最佳条件。在高盐离子环境（氯化钠浓度≥4%）中进行 EMA 处理会削弱 EMA 的抑制作用。强酸和弱酸溶液均可与 EMA 反应，改变其吸收光谱并影响 EMA 的抑制效果。由于亚致死酸化损伤，使用 EMA-qPCR 方法会低估细胞计数，在 Luria-Bertani 培养基中孵育 40 分钟可以完全消除这种误差。
我们的研究结果为 EMA-qPCR 提供了最佳的 EMA 处理、热消毒和环境条件，并证明了在不同渗透压和 pH 值环境下，该方法对活细胞计数的可行性。
优化后的 EMA 处理、热消毒和 EMA 处理环境将成功应用于 EMA-qPCR。渗透压和酸诱导损伤可以通过优化后的 EMA-qPCR 进行检测。
