Identification of mixed genotypes in Helicobacter pylori from gastric biopsy tissue by analysis or urease gene polymorphisms

Abstract

PCR-RFLP analysis of the urease A and B genes was used to investigate the frequency that Helicobacter pylori from gastric biopsy comprised genotypically distinct strains. Seventy-five strains of H. pylori from 44 subjects from four geographically diverse locations were examined and 31 distinct urease gene profiles were identified. For most cultures, the totals of the RFLP sizes were within 4% of the 2.41-kb PCR product. Five strains were genomically more complex suggesting they contained a mixture of related genotypes. The validity of the test confirmed by analysis of a known strain mixture and of single colony picks from biopsy cultures. Urease gene PCR-RFLP profiling was shown to be more sensitive than ribotyping for determining the genotypic homogeneity of H. pylori.

Development of a Scheme for Genotyping Helicobacter pylori Based on Allelic Variation in Urease Subunit Genes

“…The 383 isolates of H. pylori were collected between 1992 and 1997 from gastric or duodenal biopsy tissue taken during routine endoscopy of patients presenting with symptoms of dyspepsia (10,11,18) or from the gastric juice of healthy volunteers (21) ) and preserved at Ϫ196°C. Genomic DNA was extracted, the ureAB region was amplified, and restriction fragment length polymorphism (RFLP) analysis was performed as described previously (11,18,23). DNA fragment sizes were estimated from migration distances by using polynomial curvefitting functions (16).…”

Molecular methods for typing ofHelicobacter pyloriand their applications

“…Restriction of the 2.4‐kb fragment ( ureA and ureB genes) with Hae III or Hae III‐ Mbo I verified the diversity of H. pylori from different patients [41,42,45,46]. Mixtures of strains resulted in band sizes exceeding the expected size of 2.4 kb [42], and 7% of H. pylori clinical isolates revealed significantly larger fragments (>20%) than expected resulting in the identification of mixed genotypes [45]. Subtype variation of H. pylori before and after treatment has also been shown which was confirmed by ribotyping [46].…”

“…These genes show a high degree of homology, however single base mutations give rise to restriction site differences [39,40]. From the ureA and ureB genes of H. pylori , a 2.4‐kb product has been amplified using the supernatant from either a boiled suspension of the bacteria or purified chromosomal DNA [35,41–46]. Amplification of boiled suspensions of H. pylori is preferable because it permits PCR fingerprinting of the organism within 6 h after initial culture (Table 1).…”

Molecular methods for typing of Helicobacter pylori and their applications

“…Restriction of the 2.4-kb fragment (ureA and ureB genes) with HaeIII or HaeIII-MboI veri¢ed the diversity of H. pylori from di¡erent patients [41,42,45,46]. Mixtures of strains resulted in band sizes exceeding the expected size of 2.4 kb [42], and 7% of H. pylori clinical isolates revealed signi¢cantly larger fragments ( s 20%) than expected resulting in the identi¢cation of mixed genotypes [45]. Subtype variation of H. pylori before and after treatment has also been shown which was con¢rmed by ribotyping [46].…”