New approach to use ethidium bromide monoazide as an analytical tool

Aims: Ethidium bromide monoazide (EMA) has been determined to cause delay in DNA amplification from dead bacteria at real-time PCR. However, there is concern that the increasing EMA concentration to suppress amplification from high number of dead bacteria also affects live bacteria. The aim is to disclose a novel application of EMA for food hygienic test. Methods and Results: We performed a low-dose double EMA treatment. Live or heat-dead Enterobacter sakazakii (reclassified as Cronobacter spp.) in 10% powdered infant formula (PIF) solution was subjected to a treatment with 20 mu g ml-1 of EMA followed by a treatment with 10 mu g ml-1 of EMA without washing, and direct real-time PCR. We observed that DNA amplification from 107 cells ml-1 of dead Ent. sakazakii was completely suppressed within 50 cycles of PCR, whereas 102-103 cells ml-1 of viable cells could be detected. When a 3-h enrichment step in liquid medium was included after the first EMA treatment, live Ent. sakazakii could be detected at initial levels of 100-102 cells ml-1. We compared the low-dose double-treated EMA-PCR with the culture method using 80 samples of PIF, and completely correlative results were obtained for both methods. Conclusions: We concluded that the newly developed low-dose double-treated EMA-PCR is a very effective tool for live Ent. sakazakii detection in PIF. Significance and Impact of the Study: We focused on the specific nature of photoreactive compound that residual EMA is cancelled by irradiation. We were successful in treating bacteria with EMA in gradient concentration to increase live and dead distinction ability.