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Detection and quantification of Listeria monocytogenes by 5′‐nuclease polymerase chain reaction targeting the actA gene
Author(s) -
Oravcová K.,
Kaclíková E.,
Krascsenicsová K.,
Pangallo D.,
Brežná B.,
Siekel P.,
Kuchta T.
Publication year - 2006
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/j.1472-765x.2005.01793.x
Subject(s) - listeria monocytogenes , nuclease , polymerase chain reaction , real time polymerase chain reaction , biology , detection limit , microbiology and biotechnology , multiplex polymerase chain reaction , gene , chemistry , bacteria , chromatography , genetics
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 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. 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.