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
2010
Madic, J. | de Garam, C. Peytavin | Vingadassalon, N. | Oswald, Eric | Fach, P. | Jamet, E. | Auvray, F. | Agence Française de Sécurité Sanitaire des Aliments (AFSSA) | Institut Technique du Lait et des Produits Laitiers | Interactions hôtes-agents pathogènes [Toulouse] (IHAP) ; Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Toulouse (ENVT) ; Institut National Polytechnique (Toulouse) (Toulouse INP) ; Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National Polytechnique (Toulouse) (Toulouse INP) ; Université de Toulouse (UT)-Université de Toulouse (UT) | Université Toulouse III - Paul Sabatier (UT3) ; Université de Toulouse (UT) | Centre Hospitalier Universitaire de Toulouse (CHU Toulouse) | Ministere de l'Alimentation, de l'Agriculture et de la Peche; Association de Coordination Technique pour l'Industrie Agro-alimentaire (UMT-TERESA); ACTILAIT [241/2007]; Association Nationale de la Recherche Technique; National Interprofessional Center for the Dairy Economy (CNIEL, Paris)
International audience
Mostrar más [+] Menos [-]Inglés. 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-beta 1, eae-gamma 1, eae-epsilon and eae-theta 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.
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