title: Simplex and multiplex real-time PCR assays for the detection of flagellar (H-antigen) fliC alleles and intimin (eae) variants associated with enterohaemorrhagic Escherichia coli (EHEC) serotypes O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7.
abstract: OBJECTIVE
  To develop real-time PCR assays targeting genes encoding the flagellar antigens (fliC) and intimin subtypes (eae) associated with the five most clinically important serotypes of enterohaemorrhagic Escherichia coli (EHEC), i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7.
  
  
  RESULTS
  Primers and probes specific to fliC(H2) , fliC(H7) , fliC(H8) , fliC(H11) , fliC(H28) , eae-β1, eae-γ1, eae-ε and eae-θ were combined in simplex and multiplex 5'-nuclease PCR assays. The specificity of the assays was assessed on 201 bacterial strains and the sensitivity determined on serially diluted EHEC genomes. The developed PCR assays were found to be highly specific and detected as few as five EHEC genome equivalents per reaction. Furthermore, it was possible to detect the five major EHEC serotypes in cheese samples inoculated at concentration levels of ≤5CFU per 25g after overnight enrichment using the PCR assays.
  
  
  CONCLUSIONS
  The PCR assays developed here were found to be sensitive and specific for the reliable detection of genes encoding the flagellar antigens and intimin variants belonging to the five most clinically relevant EHEC serotypes.
  
  
  CONCLUSIONS
  Application of real-time PCR assays should improve the identification of foods contaminated by EHEC and facilitate the molecular typing of these organisms.
authors.affiliation: Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d'Etudes et de Recherches sur la Qualité des Aliments et sur les Procédés agroalimentaires (AFSSA-LERQAP), Maisons-Alfort, France.
authors.forename: J
authors.initials: J
authors.lastname: Madic
authors.forename: C
authors.initials: C
authors.lastname: Peytavin de Garam
authors.forename: N
authors.initials: N
authors.lastname: Vingadassalon
authors.forename: E
authors.initials: E
authors.lastname: Oswald
authors.forename: P
authors.initials: P
authors.lastname: Fach
authors.forename: E
authors.initials: E
authors.lastname: Jamet
authors.forename: F
authors.initials: F
authors.lastname: Auvray
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics*; D000483:Alleles; D002611:Cheese / Q000382:microbiology; D054324:Enterohemorrhagic Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D005408:Flagellin; D005516:Food Microbiology / Q000379:methods*; D058889:Molecular Typing / Q000379:methods*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D018829:Adhesins, Bacterial; D029968:Escherichia coli Proteins; C516095:FliC protein, E coli; D005408:Flagellin; C073819:eaeA protein, E coli