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A Rapid and Cost‐Efficient Technique for Simultaneous/Duplex Detection of Listeria Monocytogenes and Escherichia Coli O157:H7 Using Real Time PCR
Author(s) -
Kaynak Ahmet,
Şakalar Ergün
Publication year - 2016
Publication title -
journal of food safety
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.427
H-Index - 43
eISSN - 1745-4565
pISSN - 0149-6085
DOI - 10.1111/jfs.12254
Subject(s) - listeria monocytogenes , escherichia coli , detection limit , polymerase chain reaction , real time polymerase chain reaction , biology , duplex (building) , food safety , food industry , microbiology and biotechnology , food science , dna , bacteria , chemistry , gene , chromatography , genetics
The increasing industrial interest in easy, economical and reliable methods has led to the development and application of DNA‐based methods for the detection of microbial pathogens in food. In the present article, we describe the development of a cost‐efficient EvaGreen‐based real‐time Polymerase chain reaction (PCR) technique for simultaneous and practical detection of Listeria monocytogenes ( L. monocytogenes ) and Escherichia coli O157:H7 ( E. coli O157:H7) in the food matrix. EvaGreen‐based simplex and duplex real‐time PCR showed that specific PCR products were identified by melting curve analysis, and had a reproducible T m of 77.00 ± 0.4C for L. monocytogenes and 85.60 ± 0.2 for E. coli O157:H7.The absolute detection limit of Eva Green‐based simplex and duplex real‐time PCR was 0.0001 ng/μL for DNA of both pathogens. The relative detection limit of the simplex and duplex technique was less than 10 cells/mL for the two pathogens. Practical Applications This study illustrates a rapid, effective and cheap identification method to simultaneously monitor two foodborne pathogens in food samples. The specificity and sensitivity of this method can be used to clearly identify these two foodborne pathogens in practical food samples based on the specific genes of the species. Hence, this detection method is applicable to surveillance measures for these two foodborne pathogens in the food production chain. Moreover, this technique is used by food control laboratories in a secure way.