title: Duplex real-time PCR detection of type III effector tccP and tccP2 genes in pathogenic Escherichia coli and prevalence in raw milk cheeses.
abstract: OBJECTIVE
  To develop a duplex real-time PCR assay targeting enterohaemorrhagic Escherichia coli (EHEC) type III effector TccP/TccP2-encoding genes which are pivotal to EHEC-mediated actin cytoskeleton reorganization in human intestinal epithelial cells.
  
  
  RESULTS
  The specificity of the assay was demonstrated with DNA from EHEC reference strains and non-E. coli bacterial species. The detection limit was determined as five tccP or tccP2 copies per reaction. The assay was then evaluated on a large collection of 526 E. coli strains of human, animal, food and environmental origins. The results showed that tccP was restricted to a limited number of serotypes (i.e. O5:H(-) , O55:H7, O125:H6 and O157:H7). The tccP2 gene was present in a higher number of serotypes including the five most frequent EHEC serotypes (i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7), and a few other serotypes that caused human infections (i.e. O4:H(-) , O45:H2 and O55:H7). A minority of O26:H11 and O103:H2 strains however tested negative for tccP2, though it is not known whether the lack of tccP2 affected their pathogenic potential. Real-time PCR analysis of 400 raw milk cheeses revealed the presence of tccP and/or tccP2 genes in 19·75% of the cheese enrichment suspensions.
  
  
  CONCLUSIONS
  A highly specific and sensitive duplex real-time PCR method was developed for rapid and simultaneous detection of tccP and tccP2. Unpasteurized dairy products may be contaminated with E. coli strains carrying tccP and/or tccP2.
  
  
  CONCLUSIONS
  The developed real-time PCR assay represents a valuable alternative to conventional PCR tests and should be useful for characterization of the virulome of pathogenic E. coli strains.
authors.affiliation: ANSES, Maisons-Alfort Laboratory for Food Safety, 23 avenue du Général de Gaulle, Maisons-Alfort, France.
authors.forename: J
authors.initials: J
authors.lastname: Madic
authors.forename: C Peytavin
authors.initials: CP
authors.lastname: de Garam
authors.forename: H
authors.initials: H
authors.lastname: Brugère
authors.forename: E
authors.initials: E
authors.lastname: Loukiadis
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: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics*; D002611:Cheese / Q000382:microbiology*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D005516:Food Microbiology / Q000379:methods*; D005798:Genes, Bacterial / Q000235:genetics; D008892:Milk / Q000382:microbiology; D016133:Polymerase Chain Reaction
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C526042:TccP2 protein, E coli; C505370:tccP protein, E coli