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The aim of this study was to develop a 5′-nuclease polymerase chain reaction (PCR) for the rapid detection and quantification of Listeria monocytogenes.Specific primers and a fluorogenic probe were designed, which target a specific sequence of the actA gene encoding for a protein involved in the actin filament assembly. The PCR system was highly sensitive and specific for L. monocytogenes (inclusivity, 100%; exclusivity, 100%), which was determined using 46 L. monocytogenes and 28 non-L. monocytogenes strains. Detection limits of 104 cfu ml−1 after 35 cycles and 102 cfu ml−1 after 45 cycles were achieved by PCR in both real-time and end-point fluorescence measurement modes. Linear calibration lines were obtained in the range from 102 to 109 cfu ml−1 for three L. monocytogenes strains in real-time PCR with 45 cycles.The developed 5′-nuclease PCR of the actA gene provides a new target for the rapid detection and quantification of L. monocytogenes.In conjunction with enrichment or with an appropriate quantitative sample preparation technique, the method is suitable for food safety applications.