title: Comparison of a PCR-restriction fragment length polymorphism (PCR-RFLP) assay to pulsed-field gel electrophoresis to determine the effect of repeated subculture and prolonged storage on RFLP patterns of Shiga toxin-producing Escherichia coli O157:H7.
abstract: In this study, we compared a recently developed PCR-restriction fragment length polymorphism (PCR-RFLP) assay with pulsed-field gel electrophoresis (PFGE) using three different Shiga toxin-producing Escherichia coli (STEC) strains to understand whether repeated subculture in vitro and prolonged storage at room temperature affect the RFLP patterns of STEC. The PFGE profiles of the STEC strains changed by 1 to 8 fragments after repeated subculture and prolonged storage; one strain was no longer clonal after repeated subculture compared to the original isolate according to the Tenover criteria. In contrast, RFLP patterns obtained by PCR-RFLP were identical after repeated subculture and prolonged storage. These data clearly indicate that the PCR-RFLP assay which is based on the diversity of region V, a regulatory region of Stx-phage, was not affected by repeated subculture and prolonged storage and is a more practical and reliable method for molecular typing of STEC strains.
authors.affiliation: Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
authors.forename: Kensuke
authors.initials: K
authors.lastname: Shima
authors.forename: Yuluo
authors.initials: Y
authors.lastname: Wu
authors.forename: Norihiko
authors.initials: N
authors.lastname: Sugimoto
authors.forename: Masahiro
authors.initials: M
authors.lastname: Asakura
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Nishimura
authors.forename: Shinji
authors.initials: S
authors.lastname: Yamasaki
mesh_terms: D016521:Electrophoresis, Gel, Pulsed-Field; D019453:Escherichia coli O157 / Q000235:genetics* / Q000472:pathogenicity; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D022603:Shiga Toxins / Q000096:biosynthesis*
chemical_list: D022603:Shiga Toxins