title: Restriction-site-specific PCR as a rapid test to detect enterohemorrhagic Escherichia coli O157:H7 strains in environmental samples.
abstract: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in industrialized countries. We developed a rapid and simple test for detecting E. coli O157:H7 using a method based on restriction site polymorphisms. Restriction-site-specific PCR (RSS-PCR) involves the amplification of DNA fragments using primers based on specific restriction enzyme recognition sequences, without the use of endonucleases, to generate a set of amplicons that yield "fingerprint" patterns when resolved electrophoretically on an agarose gel. The method was evaluated in a blinded study of E. coli isolates obtained from environmental samples collected at beef cattle feedyards. The 54 isolates were all initially identified by a commonly used polyclonal antibody test as belonging to O157:H7 serotype. They were retested by anti-O157 and anti-H7 monoclonal antibody enzyme-linked immunosorbent assay (ELISA). The RSS-PCR method identified all 28 isolates that were shown to be E. coli O157:H7 by the monoclonal antibody ELISA as belonging to the O157:H7 serotype. Of the remaining 26 ELISA-confirmed non-O157:H7 strains, the method classified 25 strains as non-O157:H7. The specificity of the RSS-PCR results correlated better with the monoclonal antibody ELISA than with the polyclonal antibody latex agglutination tests. The RSS-PCR method may be a useful test to distinguish E. coli O157:H7 from a large number of E. coli isolates from environmental samples.
authors.affiliation: Infectious Diseases and Immunity Program, School of Public Health, University of California, Berkeley, California 94720, USA.
authors.forename: R
authors.initials: R
authors.lastname: Kimura
authors.forename: R E
authors.initials: RE
authors.lastname: Mandrell
authors.forename: J C
authors.initials: JC
authors.lastname: Galland
authors.forename: D
authors.initials: D
authors.lastname: Hyatt
authors.forename: L W
authors.initials: LW
authors.lastname: Riley
mesh_terms: D000821:Animal Feed; D000818:Animals; D002417:Cattle; D016172:DNA Fingerprinting; D004783:Environmental Microbiology; D004797:Enzyme-Linked Immunosorbent Assay; D004927:Escherichia coli Infections / Q000382:microbiology*; D019453:Escherichia coli O157 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012477:Salmonella enteritidis / Q000235:genetics; D012680:Sensitivity and Specificity; D012703:Serotyping