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.
Conventional plating methods for enumerating Staphylococcus aureus are time-consuming and labor-intensive. Rapid methods for specific quantification are desirable. Fluorescence in situ hybridization with filter-cultivation (FISHFC) method has been already developed to enumerate viable specific microorganisms such as Listeria monocytogenes, Clostridium perfringens and Vibrio parahaemolyticus. The purpose of this study was to develop and estimate FISHFC method for S. aureus quantitative detection in food samples. Alexa Fluor® 546-labeled oligonucleotide probe, STA68, was newly designed from the 16S rRNA gene sequences of S. aureus. STA68-conferred fluorescence was observed for S. aureus but not for any other organisms, suggesting that STA68 probe is highly specific for S. aureus. Results were achievable within 12 hours by FISHFC method, containing with 10 hours cultivation, as compared to more than 3 days required for confirmation of S. aureus by conventional plating methods. When inoculated to BPW and food samples, the numbers of viable counts determined by FISHFC method were not significantly different to those obtained by the conventional plating method (p>0.05). This result suggests that FISHFC method was preferable for the specific, rapid and accurate quantification of viable S. aureus in food.