Volume 27 Issue 10
Oct.  2014
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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
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

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

doi: 10.3967/bes2014.112
Funds:  the China Mega Project for Infectious Disease (2013ZX10004-001,2012ZX10004-215,2013ZX10004-202, and2013ZX10004804-007)
  • 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).
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    沈阳化工大学材料科学与工程学院 沈阳 110142

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

doi: 10.3967/bes2014.112
Funds:  the China Mega Project for Infectious Disease (2013ZX10004-001,2012ZX10004-215,2013ZX10004-202, and2013ZX10004804-007)

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).

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
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

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