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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 (EMABMPN-PCR). In addition, to avoid falsenegative 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 mg/ml in the preliminary experiments; subsequnently, the e#ectiveness 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 EMABMPN-PCR were almost the same as or higher than those determined using the o$cial method. Moreover, with the EMABMPN-PCR method, other Vibrio species (e.g., Vibrio harveyi) could be specifically distinguished from V. parahaemolyticus. EMABMPNPCR was shown to be a rapid and sensitive technique for counting viable V. parahaemolyticus in food and environmental samples.

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