title: Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity in identification of enterohemorrhagic Escherichia coli strains.
abstract: PCR products of 1.8 kb were generated with DNAs from all Escherichia coli H7 strains tested by using oligonucleotide primers which flank the fliC gene. Three RsaI digestion profiles of these PCR products were evident on agarose gels; the first occurred with serotype O55:H7, O157:H7, or nonmotile (NM) strains, the second occurred with serotype O1:H7 and O18:H7 strains, and the third occurred with serotype O?:H7, O19:H7, O121:H7, O88:H7, and O156:H7 strains. Despite these differences, the nucleotide sequences of the E. coli E32511 (O157:NM) and U5-41 (O1:H7) fliC genes were 97% homologous. Two PCR primer pairs synthesized on the basis of the E32511 H7 fliC sequence amplified specific DNA fragments from all E. coli H7 strains, but did not amplify DNA fragments from the other bacterial strains. The H7-specific primers were used in combination with other primers which target the Verotoxin 1(VT1) and VT2 genes and the E. coli O157:H7 eaeA gene in multiplex PCR assays. In these assays, vt and eaeA PCR products were observed with DNAs from the majority of EHEC strains and vt, eaeA, and fliC PCR products were observed with DNAs from E. coli O157:H7 or NM strains. Only eaeA PCR products were present with DNA from enteropathogenic E. coli, and only vt PCR products occurred with VT-producing E. coli which are not EHEC. The multiplex PCR assays described allow for the specific identification of E. coli O157:H7 or NM and other EHEC strains.
authors.affiliation: Animal Diseases Research Institute, Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada. gannonv@em.agr.ca
authors.forename: V P
authors.initials: VP
authors.lastname: Gannon
authors.forename: S
authors.initials: S
authors.lastname: D'Souza
authors.forename: T
authors.initials: T
authors.lastname: Graham
authors.forename: R K
authors.initials: RK
authors.lastname: King
authors.forename: K
authors.initials: K
authors.lastname: Rahn
authors.forename: S
authors.initials: S
authors.lastname: Read
mesh_terms: D018829:Adhesins, Bacterial; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000382:microbiology; D019453:Escherichia coli O157 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D029968:Escherichia coli Proteins; D005407:Flagella / Q000235:genetics*; D005408:Flagellin / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D012703:Serotyping; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2
chemical_list: D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001427:Bacterial Toxins; D002352:Carrier Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D005408:Flagellin; C073819:eaeA protein, E coli