A Rapid and Cost-Efficient Technique for Simultaneous/Duplex Detection of Listeria Monocytogenes and Escherichia Coli O157:H7 Using Real Time PCR

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 77.000.4C 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/L 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.