title: 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 Shigella 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 3.9×102 CFU/mL for S. aureus, 4.4×102 CFU/mL for L. monocytogenes, 3.0×102 CFU/mL for Salmonella, 2.5×102 CFU/mL for Shigella, 2.1×102 CFU/mL for V. parahaemolyticus, and 1.2×102 CFU/mL for E. coli. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 105 CFU/mL.
  
  
  CONCLUSIONS
  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).
authors.affiliation: Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
authors.forename: Pei Hua
authors.initials: PH
authors.lastname: Niu
authors.affiliation: Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
authors.forename: Ji
authors.initials: J
authors.lastname: Wang
authors.affiliation: Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
authors.forename: Wen Jie
authors.initials: WJ
authors.lastname: Tan
authors.affiliation: Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
authors.forename: Xue Jun
authors.initials: XJ
authors.lastname: Ma
mesh_terms: D000818:Animals; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D005516:Food Microbiology / Q000379:methods*; D008892:Milk / Q000382:microbiology; D060885:Multiplex Polymerase Chain Reaction; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity
keywords: Bacteria; Detection; Melting curve; Real-time PCR