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Keywords:

  • DNA diagnostics;
  • ethidium monoazide bromide;
  • Listeria monocytogenes;
  • real-time PCR;
  • viable but not culturable;
  • viable/dead diagnostics

Abstract

Aims:  Surface contamination by Listeria monocytogenes of gouda-like cheeses during processing represents a potential public health problem. The aim of this work was to develop novel real-time PCR diagnostics to detect the presence of viable, dead or viable but not culturable (VBNC) cells on gouda-like cheeses.

Methods and Results:  We used ethidium monoazide bromide (EMA)-PCR for direct quantification of viable and dead cells, while semiquantitative detection of culturable cells below the PCR detection limit (c. 100 CFU g−1) was obtained by combining growth and real-time PCR. We were able to quantify the fraction of >0·5% viable cells in a background of dead cells by EMA-PCR, given that the viable cell concentration was above the PCR detection limit. The combined growth and real-time PCR complemented the EMA-PCR, and enabled semiquantitative detection of low levels of culturable cells (10 and 100 CFU g−1).

Significance and Impact of the Study:  The significance of this work is that we have developed a novel concept for detection of viable and potentially infectious L. monocytogenes.