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`Molecular and immunological approaches to the separation, concentration and detection of VTEC including E. coli O157` - FS1231

A.To design and validate methods for the separation, concentration and detection of EAE+ VTEC in relevant food and environmental samples; methods to be evaluated including immunomagnetic, chromatographic and membrane grid techniques applied directly to samples or indirectly to enrichments: 1.To close and express recombinant EAE. 2.To design and validate a PCR for the specific detection of the eae gene in VTEC. 3.To develop methods for the application of the eae PCR to relevant food samples and samples produced by immunocapture. 4.To raise immunological reagents against recombinant EAE (monospecifc rabbit polyclonal and murine monoclonal antibodies). B.To define at the molecular level adhesins of EHEC and EPEC other than EAE and to develop reagents for the separation, concentration and detection of EAE- VTEC in foods: 5.To prepare a TnphoA mutant bank of EAE- VTEC strains, in order to identify surface arrayed proteins. 6.To screen mutants (from objective 5) for loss of pathogenicity and inability to adhere to tissue culture monolayers and native epithelia; and to clone the Tnphoa mutated gene (gene cluster) from these mutants. 7.To determine the sequence of the putative adhesin gene (from objective 6) and express the recombinant protein to make immunological reagents, as in part A.
Time-Scale and Cost
From: 1996

To: 1999

Cost: £272,440
Contractor / Funded Organisations
Veterinary Laboratories Agency