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

Abstract

Aims: The aim of this study was to develop a 5′‐nuclease polymerase chain reaction (PCR) for the rapid detection and quantification of Listeria monocytogenes. Methods and Results: 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. Conclusions: The developed 5′‐nuclease PCR of the actA gene provides a new target for the rapid detection and quantification of L. monocytogenes. Significance and Impact of the Study: In conjunction with enrichment or with an appropriate quantitative sample preparation technique, the method is suitable for food safety applications.

Limitation in the detection of Listeria monocytogenes in food in the presence of competing Listeria innocua

“…Quantitative real-time PCR system for the detection and quantification of L. monocytogenes targeting the actA gene developed previously in our laboratory (Oravcová et al 2006) was used in this study. Quantitative real-time PCR system for the detection and quantification of L. monocytogenes targeting the actA gene developed previously in our laboratory (Oravcová et al 2006) was used in this study.…”

Development and validation of qualitative SYBR®Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes

“…Most published PCR and qPCR assays to detect foodborne pathogens are not homogeneously validated (O’Grady et al 2008; Oravcova et al 2006; Rossmanith et al 2006). ISO 22119 (Anonymous 2011c) gives the general definitions and requirements about the use of qPCR in food microbiology but does not give performance criteria and their acceptance limits to validate a qPCR assay.…”

The challenge to quantify Listeria monocytogenes- a model leading to new aspects in molecular biological food pathogen detection

“…Such alternative methods have been described, including PCR‐based direct detection methods and pre‐enrichment procedures combined with PCR (Hein et al. 2006; Oravcová et al. 2006; Rossmanith et al.…”