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One limitation of DNA-based molecular assays is their inability to distinguish between live and dead cells. A sample treatment with propidium monoazide (PMA) before DNA amplification has been proposed to overcome this problem. The aim of this in vitro study was to test different concentrations of PMA coupled with quantitative polymerase chain reaction (qPCR) for the detection of viable Enterococcus faecalis.Viable or heat-killed suspensions of E. faecalis (10 colony-forming units/mL) were treated with PMA at 10, 50, and 100 μg/mL before DNA extraction. qPCR was performed using primers complementary for E. faecalis 16S ribosomal RNA sequence. PMA was also tested on bacteria suspensions containing different proportions of viable and dead cells. Bacterial suspensions without PMA treatment were used as positive controls.The treatment of heat-killed suspensions with PMA at different concentrations significantly reduced the DNA amplification when compared with the group without treatment (P < .0001), indicating that DNA from dead cells was not used as templates. The greatest reduction in qPCR amplification of dead cell DNA was found when 100 μg/mL PMA was used (P < .005). In mixtures containing live/dead cells, PMA allowed selective detection of viable cells.PMA was effective in inhibiting qPCR amplification from the DNA of dead cells, enabling in vitro detection and quantification of viable cells of E. faecalis.