Detection and quantification of Listeria monocytogenes by 5'-nuclease polymerase chain reaction targeting the actA gene

Abstract

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 10(4) cfu ml(-1) after 35 cycles and 10(2) 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 10(2) to 10(9) 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.