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

作者：Madic J; de Garam C Peytavin; Brugere H; Loukiadis E; Fach P; Jamet E; Auvray F^*

来源：Letters in Applied Microbiology, 2011, 52(5): 538-545.

DOI：10.1111/j.1472-765X.2011.03038.x

• enterohaemorrhagic E
• coli (EHEC)
• enteropathogenic E
• coli (EPEC)
• rapid methods
• real-time PCR
• Shiga toxin-producing E
• coli (STEC)
• tccP
• tccP2

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.

• 出版日期2011-5