Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis
doi: 10.3967/bes2014.112
-
Key words:
- Detection /
- Real-time PCR /
- Melting curve /
- Bacteria
Abstract: ObjectiveThis 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,andShigellain tube 1, Staphylococcus aureus,Vibrio parahaemolyticus, and Listeria monocytogenesin tube 2). MethodsA two-tube MCMRT-PCR assaywas 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. ResultsThe detection limit of optimized MCMRT-PCR assay was 3.9×102 CFU/mLforS. aureus, 4.4×102 CFU/mL for L. monocytogenes, 3.0×102 CFU/mL forSalmonella, 2.5×102 CFU/mL forShigella, 2.1×102 CFU/mL forV. parahaemolyticus, and 1.2×102 CFU/mL forE.coli. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 105 CFU/mL. ConclusionA 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).
Citation: | NIU Pei Hua, ZHANG Chen, WANG Ji, TAN Wen Jie, MA Xue Jun. Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis[J]. Biomedical and Environmental Sciences, 2014, 27(10): 770-778. doi: 10.3967/bes2014.112 |