Discrimination of γ-irradiated and nonirradiated Vibrio vulnificus by using real-time polymerase chain reaction

Aims: To develop a PCR strategy for Vibrio vulnificus in irradiated foods. Methods and Results: Real-time PCR was used to discriminate between DNA from gamma-irradiated and nonirradiated cells. gamma-Irradiation at 1.08 KGy and above of cell suspensions containing 1 x 10(6) CFU ml(-1) resulted in 0 CFU ml(-1). Real-time PCR was able to detect 86.6% destruction by 1.08 KGy, while ethidium bromide monoazide (EMA) real-time PCR was able to detect 93.2% destruction at this dose. With 3.0 and 5.0 KGy, EMA real-time PCR was able to detect 99.3% and 100% destruction, respectively. Conclusions: The inability to detect via PCR extensively degraded DNA resulting from gamma-irradiation can be taken as evidence of cell death. The increased ability of EMA to further reduce the detectable number of target sequences via PCR, with DNA from cells exposed to increased doses of gamma-irradiation, can be considered to reflect the accompanying increase in membrane damage which allows EMA to penetrate the cells. Significance and impact of the study: This is the first study that has made use of the PCR to discriminate between gamma-irradiated and nonirradiated bacterial cells and has led to insights regarding the ability of the PCR to discriminate between nonirradiated and gamma-irradiation destroyed cells.