Detection of Viable Escherichia coli O157:H7 by Propidium Monoazide Real-time PCR

Introduction: Escherichia coli O157:H7 associated with food has caused many serious public health problems in recent years. However, only viable cells of this pathogen can cause infections, and false-positive detection caused by dead cells, can lead to unnecessary product recalls. 

Purpose:   The objective of this study was to develop and optimize a method that combines propidium monoazide (PMA) staining with real-time PCR to detect only viable cells of E. coli O157:H7.  PMA is a dye that can penetrate dead cells and bind to cellular DNA, preventing its amplification via a subsequent PCR.  Compared with ethidium monoazide (EMA), another DNA-binding, PMA has been reported to exert less influence on DNA amplification from viable cells.

Methods:   Each of three strains of E. coli O157:H7 (505B, G5310 and C7927) was prepared separately and serially diluted to generate cell suspensions ranging from 10 to 10⁸ CFU/ml.  Dead cells were obtained by heating the suspensions at 85 °C for 35 min. Suspensions were then treated or untreated with PMA.  DNA was extracted and amplified by TaqMan® real-time PCR targeting the uidA gene to detect only viable E. coli O157:H7 cells.

Results:   A 5-min treatment with 50 mM PMA on ice was the most effective method to bind DNA from 10⁸ CFU/ml dead cells.  PMA-real-time PCR assay could detect as low as 10² CFU/ml viable E. coli O157:H7 in live-cell suspensions.  This assay could detect 10⁶ CFU/ml of viable cells when dead cells ranging from 10 to 10⁶CFU/ml were present.  Studies detecting lower concentrations of viable cells in the presence of dead cells and in food are ongoing.

Significance:   In conclusion, the PMA-real-time PCR assay can effectively prevent amplification of DNA in dead cells of E. coli O157:H7 and differentiate viable from dead cells.