The present study compared different concentrations of propidium monoazide (PMA), time of exposure to light and different light intensities to determine the optimal conditions for the quantification of viable Escherichia coli in cell suspension and in food matrix. The influence of cell density and the effectiveness of PMA in viable but non-culturable (VBNC) E. coli cells were evaluated and also applied in food matrix. For that purpose, different concentrations of PMA (20 μM, 40 μM, 50 μM, 60 μM and 80 μM) under different times of exposure (5 min, 10 min, 15 min, 20 min and 30 min) to lights of different intensities (500 W and 650 W) were evaluated. After determining the optimal conditions, the PMA-qPCR methods were applied to different compositions of live and heat-killed E. coli suspensions (v:v; 0:1; 1:0; 1:1) in concentrations ranging from 3 Log to 7 Log CFU/mL. The same dilutions were prepared with E. coli in VBNC state and applied in food matrix. The results obtained from qPCR, PMA-qPCR and plate counts were compared. The results suggested that a PMA treatment of 50 μM PMA for 15 min under 650 W light intensity was optimal under our conditions. For E. coli cell suspensions, the amplification of heat-killed cells was inhibited greatly by PMA when concentrations were ≤ 5 Log CFU/mL. For the samples of oyster inoculated with heat-killed cells, E. coli was not detected by PMA-qPCR in concentrations ≤4 Log CFU/g. Regarding the results with VBNC state, we considered the PMA-qPCR method to be applicable for enumerating E. coli VBNC cells in oyster samples. Based on our findings, we further recommend the use of PMA-qPCR with the aim of reducing the amplification of dead cells for improving its performance, since false-positives could still occur depending on the level of E. coli in the sample. The application of the PMA-qPCR for quantification of bacteria, compared to the use of culture-dependent methods, is quite promising. However, further studies are recommended, especially using different food matrices.

本研究比较了不同浓度的单叠氮化丙锭（PMA），暴露于光的时间和不同的光强度，以确定定量分析细胞悬浮液和食物基质中活大肠杆菌的最佳条件。评估了细胞密度和PMA在活的但不可培养的（VBNC）大肠杆菌细胞中的影响，并将其应用于食品基质中。为此，在不同强度的光下（5分钟，10分钟，15分钟，20分钟和30分钟）的不同曝光时间（5分钟，10分钟，15分钟，20分钟和30分钟），使用不同浓度的PMA（20μM，40μM，50μM，60μM和80μM）评估（500 W和650 W）。在确定最佳条件后，将PMA-qPCR方法应用于不同的鲜活和热灭活成分大肠杆菌悬浮液（v：v; 0：1; 1：0; 1：1）的浓度范围为3 Log至7 Log CFU / mL。用VBNC状态的大肠杆菌制备相同的稀释液，并将其稀释在食品基质中。比较了从qPCR，PMA-qPCR和板数获得的结果。结果表明，在我们的条件下，以650 W的光强度对50μMPMA进行15分钟的PMA处理是最佳的。对于大肠杆菌细胞悬液，当浓度≤5 Log CFU / mL时，PMA会极大地抑制热杀死细胞的扩增。对于用热灭活细胞接种的牡蛎样品，大肠杆菌浓度≤4Log CFU / g的PMA-qPCR未检测到任何SNP。关于VBNC状态的结果，我们认为PMA-qPCR方法适用于枚举牡蛎样品中的大肠杆菌VBNC细胞。根据我们的发现，我们进一步建议使用PMA-qPCR，其目的是减少死细胞的扩增以提高其性能，因为取决于样品中大肠杆菌的水平，假阳性仍然可能发生。与使用依赖于培养物的方法相比，将PMA-qPCR应用于细菌定量的方法非常有前途。但是，建议进一步研究，尤其是使用不同的食物基质时。