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FP5

ANTRES Résumé de rapport

Project ID: ICA4-CT-2001-10014
Financé au titre de: FP5-INCO 2
Pays: Italy

Development of a DNA-based method for rapid and inexpensive identification of Escherichia coli

In consideration that the commercial biochemical identification systems for E. coli are expensive and time-consuming, and therefore poorly suitable for analysing a large number of samples to be investigated in monitoring screening, a method for rapid, inexpensive and reliable identification of E. coli by means of filter DNA hybridisation was developed.

Two different targets were selected to develop species-specific DNA probes for E. coli: the lacZ gene, encoding galactosidase, and the uidA gene, encoding glucuronidase, which has already been used to develop species-specific DNA probes for E. coli (McDaniels et al., Appl. Environ. Microbiol., 1996, 62:3350-3354). The hybridization probe for the lacZ gene was generated by PCR amplification of a 340-bp segment of the lacZ gene from E. coli CCUG24T, using primers Bgal-fwd (5 -TCTGGAAGATCAGGAATATGTGG) and Bgal-rev (5 -ATAGAGATTCGGGATTTCGGC). The hybridization probe for the uidA gene was generated by PCR amplification of an 882-bp segment of the uidA gene from E. coli CCUG24T, using primers uidA-fwd (5-TGGGCATTCAGTCTGGATC) and uidA-rev (5 -GCACCATCAGCACGTTATC).

The specificity of each probe was tested in a set of preliminary colony blot experiments using reference strains of E. coli, Klebsiella spp., Serratia spp., Citrobacter spp., Shigella spp., Providencia spp., Kluyvera spp. and Salmonella enterica. In these experiments the uidA probe was found to be more specific that the lacZ probe, and for this reason it was selected for further development. To validate the uidA probe, it was tested on a sub sample of 466 isolates collected during the pilot study, and results, obtained in a blinded fashion, were compared with those obtained with the conventional API 20E biochemical identification system.

The two methods were in agreement with all the isolates, with the exception of 3 isolates which were identified as E. coli by the API 20E system but were not recognized by the uidA probe, and of 2 isolates which gave a positive hybridization signal with the uidA probe but couldn�t be identified with certainty with the API20E system. These results are overall consistent with previous observations indicating that 97 to 100% of E. coli strains carry the uidA gene (McDaniels et al., Appl. Environ. Microbiol., 1996, 62:3350-3354).

According to these results, the colony blot protocol based on the uidA probe appeared to be a sensitive and specific method for identification of E. coli isolates. In comparison with conventional biochemical testing, this method is less time-consuming and much cheaper (less than 1 Euro vs. 10 Euro per tested isolate). For these reasons, it was decided to adopt this method for E. coli identification in the baseline ARS study.

Informations connexes

Contact

Gian Maria ROSSOLINI, (Full professor)
Tél.: +39-0577233326
Fax: +39-0577233325
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