Development and Application of a One‐Tube Multiplex Real‐Time PCR with Melting Curve Analysis for Simultaneous Detection of Five Foodborne Pathogens in Food Samples

Abstract

Abstract This study was aimed at developing a one‐tube multiplex real‐time PCR system for simultaneous detection of five major foodborne pathogens ( Staphylococcus aureus , Listeria monocytogenes , Salmonella enterica , Vibrio parahaemolyticus and Shigella spp.) in food samples. The newly developed multiplex real‐time PCR was performed using fluorescent dye EvaGreen and melting curve analysis for pathogen discrimination. The specificity of the PCR method was evaluated by testing with DNA extracted from different strains. The detection limits of genomic DNA per reaction for the five pathogens ranged from 0.39 pg ( Shigella spp.) to 40 pg ( Listeria monocytogenes) . Sensitivity and specificity of this multiplex real‐time PCR were further confirmed using artificially and naturally contaminated samples. The newly developed multiplex real‐time PCR was useful for rapid, specific and cost‐effective detection of the five target pathogens in contaminated food samples. Practical Applications The newly developed multiplex real‐time PCR assay could detect five major foodborne pathogens ( Staphylococcus aureus , Listeria monocytogenes , Salmonella enterica , Vibrio parahaemolyticus and Shigella spp.) in food samples in a single reaction tube. This method is simple, cost‐effective, specific, sensitive and is capable of high‐throughput detection for five foodborne pathogens simultaneously. Therefore, it has a potential application as a useful tool in food testing laboratories for quick identification of foodborne pathogens.