Evaluation of a Rapid Test for Determination of Most Probable Number of Viable Vibrio parahaemolyticus by Using Etidium Monoazide

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.