Enumeration of viable Listeria monocytogenes cells by real-time PCR with propidium monoazide and ethidium monoazide in the presence of dead cells

Abstract

Propidium monoazide (PMA) and ethidium monoazide were used for enumeration of viable Listeria monocytogenes cells in the presence of dead cells. PMA had no antimicrobial effect on L. monocytogenes. Viable cell counts were linearly related to real-time PCR threshold cycle values for PMA-treated cells from planktonic and biofilm sources over a 4-log range.

FIG. 1.

Effects of PMA and EMA on DNA amplification from viable and dead cells of L. monocytogenes. (A and B) Viable cell density was determined following 5 min of incubation in the dark (A) and after 5 min of exposure to light (B). (C and D) Effects of PMA and EMA on DNA amplification from viable cells (C) and heat-killed cells (D). The bars indicate the mean values, and the error bars indicate the standard deviations (n = 3). Different letters indicate a significant difference (P < 0.05).

FIG. 2.

Standard curves for quantifying viable L. monocytogenes cells in a viable cell-dead cell mixture (A) and in stressed biofilms (B). The solid standard line and the linear regression coefficient factor (R²) were generated using the solid triangles. The open squares are data points not included in the linear regression analysis.