Purpose: The purpose of this study was to develop and optimize a rapid, PCR-based detection method combined with DNA photo-labeling able to differentiate live and dead E. coli O157:H7.
Methods: The procedure was optimized for full neutralization of dead cells, while maintaining amplification of live cells. Both live and dead E. coli O157:H7 (a farm isolate) culture samples were treated with or without DNA photo-labeling dye ethidium monoazide (EMA). Samples were then exposed to LED light and analyzed using multiplex PCR (mPCR) and quantitative PCR (qPCR).
Results: Under the optimized DNA photo-labeling condition of five-minute incubation with 25 µM EMA followed by five-minute of high-intensity LED light exposure, DNA from dead cells was neutralized and PCR amplification occurred only with DNA from live cells. Live cells were successfully differentiated from dead cells with a detection limit of 105 CFU/ml in mPCR. In qPCR where 106 to 108CFU/ml were tested, it was noted that the lower cell numbers gave better differentiation between dead and live cells.
Significance: This data suggested that DNA photo-labeling combined with PCR-based detection methods can differentiate live and dead E. coli O157:H7.