INSERTION ELEMENT IS3-BASED PCR METHOD FOR SUBTYPING ESCHERICHIA-COLIO157-H7

An Escherichia coli O157:H7 subtyping method based on PCR amplificatio n of variable DNA sequences between the repetitive element IS3 was dev eloped. Template DNA was prepared by boiling cells in Chelex. Two sepa rate IS3 PCR amplifications were performed for each isolate: one with a single primer (primer IS3A) and one with two primers (primers IS3A a nd IS3B). The IS3 PCR subtyping method was applied to 35 epidemiologic ally related and unrelated E. coli O157:H7 isolates that had been prev iously characterized by pulsed-field gel electrophoresis (PFGE). PFGE identified 25 different subtypes (difference of one or more hands), PC R with single primer IS3A and primer pair IS3A-IS3B identified 6 and 1 4 different subtypes, respectively. By combining the results of the tw o PCR amplifications, 15 different IS3 PCR subtypes were identified. W hile not as sensitive as PFGE, IS3 PCR subtyping grouped all outbreak- related isolates. IS3 PCR banding patterns were reproducible between a mplifications and between subcultures. IS3 PCR could serve as a simple , rapid screening method for the identification of unrelated E. coli O 157:H7 isolates.