Insertion Element IS 3 -Based PCR Method for Subtyping Escherichia coli O157:H7

Abstract

An Escherichia coli O157:H7 subtyping method based on PCR amplification of variable DNA sequences between the repetitive element IS3 was developed. Template DNA was prepared by boiling cells in Chelex. Two separate IS3 PCR amplifications were performed for each isolate: one with a single primer (primer IS3A) and one with two primers (primers IS3A and IS3B). The IS3 PCR subtyping method was applied to 35 epidemiologically related and unrelated E. coli O157:H7 isolates that had been previously characterized by pulsed-field gel electrophoresis (PFGE). PFGE identified 25 different subtypes (difference of one or more bands). PCR with single primer IS3A and primer pair IS3A-IS3B identified 6 and 14 different subtypes, respectively. By combining the results of the two PCR amplifications, 15 different IS3 PCR subtypes were identified. While not as sensitive as PFGE, IS3 PCR subtyping grouped all outbreak-related isolates. IS3 PCR banding patterns were reproducible between amplifications and between subcultures. IS3 PCR could serve as a simple, rapid screening method for the identification of unrelated E. coli O157:H7 isolates.

Comparison of PCR-based methods for typing Escherichia coli

“…These poor results are in contrast to other reports [12,14]. The reason for this could be the different range of fragment sizes present when analyzing the fingerprints by use of DNA sequencing machines, or the fact that the method was originally actually devised for enteropathogenic strains.…”

“…The typing methods investigated were RAPD (primers: M13, DAF4, 1247), rep‐PCR (primers: ERIC‐2, IS3A in combination with IS3B), and AFLP ( Eco RI‐0 + Mse I‐TA). The different PCR reactions were essentially performed as described previously [7,9,10,12–15]. The oligonucleotides, their DNA sequences and the PCR annealing temperatures ( T ann ) are listed in Table 1.…”

“…The aim of this study was three‐fold. First, different published protocols for typing by means of RAPD, rep‐PCR and fluorescent amplified fragment length polymorphism (AFLP) were compared with PFGE, using a small panel of epidemiologically unrelated strains [7,9,10,12–15]. Second, an efficient method for analysis of AFLP fingerprints was sought, as well as a sensible, though preliminary, estimate of a cut‐off value to discriminate genotypically related and non‐related isolates.…”

Molecular diversity of Escherichia coli in the human gut: New ecological evidence supporting the role of adherent-invasive E. coli (AIEC) in Crohnʼs disease

“…The first step in analyzing E. coli clonality was performed using the Insertion Element IS3‐Based PCR subtyping method and primers IS3A and IS3B together, as described previously 29. Rep‐PCR profiles were compared using the GelComparII software (Applied Maths, Belgium).…”

RAPID AND EFFICIENT GENERATION OF PCR TEMPLATES FROM ESCHERICHIA COLI, SACCHAROMYCES CEREVISIAE AND ORYZA SATIVA USING A MICROWAVE AND BY BOILING

“…Polymerase chain reaction (PCR) is a powerful technique with widespread applications in molecular biology. Many researchers have come up with many ways of preparing PCR templates and plasmids (Hultner and Cleaver 1994;Wang et al 1995;Ferreira and Louise 1996;Yeadon and Catcheside 1996;Thompson et al 1998;Jimenez et al 2000), many of which require special chemicals, such as detergent and surfactants, and are multistep. As current research's high-throughput nature requires robust template preparation methods, we have developed simple methods using a microwave and by boiling to prepare templates from Escherichia coli , Saccharomyces cerevisiae and Oryza sativa , as well as other organisms (Table 1).…”