NUCLEOTIDE-SEQUENCE ANALYSIS OF ENTEROPATHOGENIC ESCHERICHIA-COLI (EPEC) ADHERENCE FACTOR PROBE AND DEVELOPMENT OF PCR FOR RAPID DETECTION OF EPEC HARBORING VIRULENCE PLASMIDS

The 1-kb BamHI-SalI fragment from plasmid pMAR2 termed the enteropatho genic Escherichia coli (EPEC) adherence factor (EAF) probe was cloned in pUC19 and pIUS. The nucleotide sequence of this fragment was determ ined, and a set of primers was designed to amplify a 397-bp region ass ociated with pMAR2 by PCR. An analysis of the whole EAF sequence with database libraries indicated no significant homology to any known gene s. However, between bases 701 and 787 of the fragment, an 82.8% homolo gy between the EAF and the insertion Sequence IS630 of Shigella sonnei exists. The results of PCR with primers of the EAF sequence demonstra ted that all of the 151 EAF probe-positive EPEC strains with localized adherence to HEp-2 cells yielded positive EAF PCR results. In contras t, none of the 277 EAF probe-negative strains reacted to the EAF PCR. In addition, the PCR assay was successfully used to generate vector-fr ee digoxigenin-labeled EAF fragments that gave valid results in colony blot hybridization assays. The EAF PCR appears to be a specific and e fficient method for the detection of EPEC strains carrying the EAF pla smids.