Rapid Detection of Live E. coli O157:H7 by PMA-qPCR Method

In this work, a method to detect live E. coli O157:H7 was established using propidium monoazide (PMA) coupled with quantitative polymerase chain reaction (qPCR). The minimum inhibitory concentration against DNA amplification from dead bacteria was 10 μg/mL, and PMA did not inhibit DNA application from live bacteria at concentrations equal to or lower than 20 μg/mL. Covalent cross-linking of PMA with DNA was induced by strong light illumination for 5 min and meanwhile, free PMA was completely inactivated, resulting in the avoidance of false negative results. When the live to dead bacteria ratio was more than 1%, PMA pretreatment allowed the elimination of DNA interference from thermally inactivated bacteria and therefore live bacteria could be quantifi ed.