Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis.

Abstract Objective This study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay (MCMRT-PCR) for the simultaneous detection of six common foodborne pathogenic bacteria( diarrhoeagenic Escherichia coli, Salmonella , and Shigellain tube 1, Staphylococcus aureus, Vibrio parahaemolyticus, and Listeria monocytogenes in tube 2). Methods A two-tube MCMRT-PCR assay was performed on 7900HT Fast Real-Time PCR System (Applied Biosystems, USA). Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated. Results The detection limit of optimized MCMRT-PCR assay was 3.9×10 2 CFU/mL for S. aureus , 4.4×10 2 CFU/mL for L. monocytogenes , 3.0×10 2 CFU/mL for Salmonella , 2.5×10 2 CFU/mL for Shigella, 2.1×10 2 CFU/mL for V. parahaemolyticus , and 1.2×10 2 CFU/mL for E. coli . The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 10 5 CFU/mL. Conclusion A two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteriawith an intended application in provincial Centers for Diseases Control and Prevention (CDC).