Polymerase Chain Reaction Assay Coupled with Fluorescence Detection on Microwell Plates for Listeria monocytogenes in Foods

Abstract

This study evaluates a polymerase chain-reaction assay coupled with fluorescence detection (PCR-FD) in microwell plates for Listeria monocytogenes in dairy and food samples. Guanidinium thiocyanate/silica–extracted cultures and milk and dairy-food samples were used as templates for a PCR assay in microwell plates, with a primer pair that amplifies a 221 bp segment of the internal transcribed spacer (ITS), in the presence of 0.5 μg/ml of ethidium bromide. Fluorescence of the PCR products was measured with a CytoFluor 2300 fluorescence measurement system (Millipore Corporation, Bedford, MA). The lowest level of detection of the assay was 10 to 100 CFU. A total of 326 food samples were tested, both by culture and by PCR-FD. The overall sensitivity of the PCR-FD assay was 95.2% and the specificity was 89.9%. Positive and negative predictive values were 74.8% and 99.4%, respectively. Based on the results obtained in this study it appears that the PCR-FD assay described here can be useful for screening a large number of milk and dairy-food samples for contamination by Listeria monocytogenes.