- Top of page
- Materials and methods
- Strains and culture conditions
- Spiking experiments
- EMA treatment
- DNA purification
- Real-time PCR amplification
- Analyses of the real-time EMA-PCR results
- Results and discussion
- DNA purification directly from the cheese matrix
- Separate detection of viable and dead L. monocytogenes
- Potential natural contamination by L. monocytogenes
- Quantitative detection of L. monocytogenes on a cheese matrix by combined growth and real-time PCR
- Future perspectives on the quantitative diagnostics of L. monocytogenes
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.