Use of the Restriction Enzyme EcoRI for Pulsed-Field Gel Electrophoretic Analysis of Helicobacter pylori

Abstract

Pulsed-field gel electrophoretic (PFGE) analysis of Helicobacter pylori isolates is not commonly employed because of the inability to compare the typing with other typing systems. We adapted the PFGE analysis for H. pylori by using EcoRI and slightly modified our laboratory methods to improve the typing of isolates (typeability was 97%).Helicobacter pylori is associated with upper gastrointestinal diseases (1,3,4,5). Pulsed-field gel electrophoresis (PFGE) is usually used for typing bacteria for epidemiologic purposes. However, PFGE analysis of H. pylori isolates (2,6,7,8) is not commonly used, because of the inability to compare the typing with other typing systems (2). Our laboratory previously investigated using PFGE analysis for clinically isolated bacteria (9). However, this method was not effective for the analysis of H. pylori strains. To improve typing, we selected the restriction enzyme EcoRI to cut the chromosomal DNA and also modified our laboratory methods.A total of 71 isolates of H. pylori were collected, which were obtained from 31 patients. If H. pylori was isolated, one to four individual colonies were cultured for 4 to 7 days on agar plates for isolation (Eiken Chemical, Tokyo, Japan) in a gas mixture of 85% N 2 -5% O 2 -10% CO 2 at 35°C. Colonies were purified by passage on brucella agar plates (containing 10% horse serum), harvested from the plate by scraping, and suspended in 10% glycerol for storage at Ϫ80°C.We performed PFGE analysis as follows. H. pylori strains harvested from plates by scraping were suspended in 100 ml of brucella broth (Difco, Detroit, Mich.) supplemented with 10% horse serum (preheated to 56°C for 30 min) and cultured with shaking for 4 to 7 days at 35°C in a gas mixture (85% N 2 -5% O 2 -10% CO 2 ). The organisms were harvested by centrifugation, washed in a saline-EDTA solution (0.15 M NaCl, 10 mM EDTA, pH 8.0), and resuspended in Pett IV solution (1 M NaCl, 10 mM EDTA, pH 8.0). An equal volume of melted 2.0% low-melting-point agarose (Low Melt Preparative Grade; Bio-Rad Laboratories, Hercules, Calif.) was added to this suspension. The mixture was poured into an insert and chilled at 4°C for 20 min. The mixture was then treated at 37°C with 50 mg of lysozyme (Seikagaku, Tokyo, Japan)/ml in a lysis solution (1 M NaCl, 0.1 M EDTA [pH 8.0], 10 mM Tris-HCl, 0.5% Brij 58, 0.2% sodium deoxycholate, 0.5% Sarkosyl). After 1 h, the lysis solution was decanted and replaced with 1 mg of proteinase K (Wako Pure Chemical Industries, Osaka, Japan)/ml in ES solution (0.25 M EDTA [pH 8.0], 1% Sarkosyl) at 50°C for 48 h. The ES solution was decanted, and the plugs were placed in TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0]) containing 1 mM phenylmethylsulfonyl fluoride at room temperature for 4 h. The plugs were washed three times in TE buffer for 10 min at room temperature. For restriction endonuclease digestion, the plugs were incubated in 10 mM Tris-HCl-0.1 mM EDTA solution for 20 min at room temperature. This solution was decanted, and the plugs were incubated in enzyme...

Characterization of Helicobacter pylori Bacteriophage KHP30

“…Samples for pulsed-field gel electrophoresis (PFGE) were prepared from H. pylori strains 3401 and NY43 (ca. 1.7 ϫ 10 9 cells/ml), as described elsewhere (48). A bacteriophage lambda ladder PFG marker (New England BioLabs, Beverly, MA) was included in this analysis as a molecular weight standard.…”

Genotyping of the Helicobacter pylori cagA Gene Isolated From Gastric Biopsies in Shiraz, Southern Iran: A PCR-RFLP and Sequence Analysis Approach

“…Various methods have been employed for H. pylori genotyping, such as pulsed-field gel electrophoresis (PFGE), PCR-based randomly amplified polymorphic DNA (RAPD) fingerprinting, and hybridization with specific probes ( 8 - 10 ). By using restriction enzyme EcoRI and the PFGE method, a 97% improvement in H. pylori genotyping has been described ( 11 ). However, the PFGE method is time-consuming and requires a unique apparatus to handle a large number of clinical specimens.…”