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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
Author(s) -
Madic J.,
Peytavin de Garam C.,
Vingadassalon N.,
Oswald E.,
Fach P.,
Jamet E.,
Auvray F.
Publication year - 2010
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2010.04798.x
Subject(s) - intimin , biology , serotype , escherichia coli , microbiology and biotechnology , multiplex , multiplex polymerase chain reaction , virology , antigen , polymerase chain reaction , gene , enterobacteriaceae , genetics
Abstract Aims: 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. Methods and 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 ≤5 CFU per 25 g 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. Significance and Impact of the Study: Application of real‐time PCR assays should improve the identification of foods contaminated by EHEC and facilitate the molecular typing of these organisms.