Detection of viable and dead Listeria monocytogenes on gouda-like cheeses by real-time PCR

ABSTRACT K. RUDI, K. NATERSTAD, S.M. DROMTORP AND H. HOLO. 2005. Aims: Surface contamination by Listeria monocytogenes of gouda-like cheeses during processing represents apotential public health problem. The aim of this work was to develop novel real-time PCR diagnostics to detect thepresence 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 viableand 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 >0AE5% viable cellsin a background of dead cells by EMA-PCR, given that the viable cell concentration was above the PCR detectionlimit. The combined growth and real-time PCR complemented the EMA-PCR, and enabled semiquantitativedetection 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 conceptfor detection of viable and potentially infectious L. monocytogenes.Keywords: DNA diagnostics, ethidium monoazide bromide, Listeria monocytogenes, real-time PCR, viable but notculturable, viable/dead diagnostics.