Quantification of Listeria monocytogenes in salads by real time quantitative PCR

A real time quantitative PCR (RTQ-PCR) was carried out purifying DNA extracts of Listeria monocytogenes using a High Pure Listeria Sample Preparation Kit and quantifying in a LightCycler system with hybridisation probes. A standard curve was constructed with serial dilutions. A range linear relationship, from 10 to 105 L. monocytogenes colony forming units (CFU), was observed between threshold cycle (Ct) and logarithmic concentration of the serial dilutions. The assay was linear in a range from 10 to 105 L. monocytogenes CFU and the coefficient of determination (r2) was > 0.98. RTQ-PCR presented an efficiency of > 85%. The accuracy of the PCR-based assay, expressed as % bias, ranged from 9% to 26% and the precision, expressed as % CV, ranged 9–22%. Intraday and interday variabilities were studied at 102 CFU/g and resulted in 12% and 14%, respectively. The proposed RTQ-PCR method and classical cultural methods were applied to analyse 77 salads from restaurants in Valencia (Spain). All culture positive samples were also RTQ-PCR positive.