Differentiation of Live and Dead Escherichia coli O157:H7 Using a PCR-based Method Combined with DNA Photo Labeling

Introduction:  The CDC estimates Shiga toxin-producing Escherichia coli (STEC) causes over 265,000 infections and 30 deaths each year in the United States.  Escherichia coli O157:H7 is the most common STEC serotype, being responsible for approximately 36% of those illnesses. While only viable E. coli O157:H7 cells can cause illness, presence of dead cells can result in false-positive results in many detection methods. DNA photo-labeling only neutralizes DNA from dead cells and, thus can selectively amplify DNA from live cells.

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.