Restriction-site-specific PCR as a rapid test to detect enterohemorrhagic Escherichia coli O157 : H7 strains in environmental samples

Enterohemorrhagic Escherichia call (EHEC) O157:H7 is an important food-born e pathogen in industrialized countries. We developed a rapid and simple tes t for detecting E. coli O157:H7 using a method based on restriction site po lymorphisms. Restriction-site-specific PCR (RSS-PCR) involves the amplifica tion of DNA fragments using primers based on specific restriction enzyme re cognition sequences, without the use of endonucleases, to generate a set of amplicons that yield "fingerprint" patterns when resolved electrophoretica lly on an agarose gel. The method was evaluated in a blinded study of E. co li isolates obtained from environmental samples collected at beef cattle fe edyards. The 54 isolates were all initially identified by a commonly used p olyclonal antibody test as belonging to O157:H7 serotype. They were reteste d by anti-O157 and anti-arl monoclonal antibody enzyme-linked immunosorbent assay (ELISA). The RSS-PCR method identified all 28 isolates that were sho wn to be E. coli O157:H7 by the monoclonal antibody ELISA as belonging to t he O157:H7 serotype. Of the remaining 26 ELISA-confirmed non-O157:H7 strain s, the method classified 25 strains as non-O157:H7. The specificity of the RSS-PCR results correlated better with the monoclonal antibody ELISA than w ith the polyclonal antibody latex agglutination tests. The RSS-PCR method m ay be a useful test to distinguish E. coli O157:H7 from a large number of E . coli isolates from environmental samples.