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

• Published in: 生物医学与环境科学：英文版 2014, Vol.27 (10), p.770-778
• Main Author: NIU Pei Hua ZHANG Chen WANG Ji TAN Wen Jie MA Xue Jun
• Format: Article
• Language: English
• Subjects: 双管法; 同时检测; 多重; 实时PCR; 曲线分析; 熔解曲线; 金黄色葡萄球菌; 食源性致病菌
• ISSN: 0895-3988; 2214-0190

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 5higella in 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 3.9x102 CFU/mLfor S. aureus, 4.4x102 CFU/mL for L. monocytogenes, 3.0x102 CFU/mL for Salmonella, 2.5x102 CFU/mL for Shigella, 2.1x102 CFU/mL for V. parahaemolyticus, and 1.2x102 CFU/mL for E. coll. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 10s 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 bacteria with an intended application in provincial Centers for Diseases Control and Prevention （CDC）.