抄録
Ethidium monoazide (EMA) has been used for selective polymerase chain reaction (PCR) amplification of DNA extracted from viable bacterial cells. However, its use is very limited; further, it is less frequently applied for quantitative purposes, for example, bacterial counting. In this study, we evaluated the most probable number (MPN)-PCR method using the species-specific toxR as the target gene after EMA treatment, for rapid quantitative determination of viable Vibrio parahaemolyticus (EMA+MPN-PCR). In addition, to avoid false-negative results attributable to PCR inhibition owing to carryover contamination, we added the positive-control template DNA (PCT) to each reaction tube. The optimal concentration of EMA for V. parahaemolyticus cells was determined to be 5 μg/ml in the preliminary experiments; subsequnently, the effectiveness of EMA was confirmed using simulated food samples spiked with viable or dead V. parahaemolyticus cells. The results of the experiments with 38 seafood and 8 seawater samples were as follows: MPN values determined using EMA+MPN-PCR were almost the same as or higher than those determined using the official method. Moreover, with the EMA+MPN-PCR method, other Vibrio species (e.g., Vibrio harveyi) could be specifically distinguished from V. parahaemolyticus. EMA+MPN-PCR was shown to be a rapid and sensitive technique for counting viable V. parahaemolyticus in food and environmental samples.
