Duplex real-time PCR detection of type III effector tccP and tccP2 genes in pathogenic Escherichia coli and prevalence in raw milk cheeses

Autor: Madic, J., de Garam, C. Peytavin, Brugère, Hubert, Loukiadis, Estelle, Fach, P., Jamet, E., Auvray, F.
Přispěvatelé: Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), 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 Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Ministere de l'Alimentation, de l'Agriculture et de la Peche, Association de Coordination Technique pour l'Industrie Agro-alimentaire (UMT-TERESA), Transversalite AFSSA-INRA, ACTILAIT, Association Nationale de la Recherche Technique (ANRT), National Interprofessional Center for the Dairy Economy (CNIEL, Paris), [CIFRE 241/2007]
Jazyk: angličtina
Rok vydání: 2011
Předmět:
Zdroj: Letters in Applied Microbiology
Letters in Applied Microbiology, Wiley, 2011, 52 (5), pp.538-545. ⟨10.1111/j.1472-765X.2011.03038.x⟩
ISSN: 0266-8254
1472-765X
DOI: 10.1111/j.1472-765X.2011.03038.x⟩
Popis: International audience; Aims: 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. Methods and 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 center dot 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. Significance and Impact of the Study: 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.
Databáze: OpenAIRE
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