Determination of mutations of the 23S rRNA gene of Helicobacter pylori by allele specific primer-polymerase chain reaction method

Abstract

Background and aims: Susceptibility to clarithromycin of Helicobacter pylori (H. pylori) is caused by single nucleotide polymorphisms (SNPs) of the 23SrRNA gene. Allele specific primer-polymerase chain reaction (ASP-PCR) is one of the methods for determining SNPs, which can measure SNPs easily within a short period by PCR amplification alone without digestion with restriction enzymes. The aim of the present study was to develop the ASP-PCR assay for determining SNPs at positions 2,142 and 2,143 of the 23S rRNA gene of H. pylori.

Methods: In total, 112 patients with H. pylori infection based on positive results of a rapid urease test (RUT) were enrolled in the study. Thirty-five had failed to eradicate H. pylori by a clarithromycin-based regimen. DNA was extracted from the RUT-positive gastric tissue samples. SNPs from adenine to guanine at positions 2,142 and 2,143 of the 23S rRNA of H. pylori (A2,142G and A2,143G) were determined by the ASP-PCR method. Minimum inhibitory concentrations (MICs) of clarithromycin for H. pylori were also measured.

Results: Forty-nine of 112 patients were infected with wild-type strains of H. pylori. Thirty-nine patients were infected with strains with A2,143G mutations. Twenty-three patients were infected with both wild-type strains and those with A2,143G mutations. Only one patient was infected with the strain with A2,142G mutation. H. pylori strains with A2,143G or A2,142G mutation had significantly higher MICs for clarithromycin.

Conclusion: The ASP-PCR assay for 23S rRNA mutation of H. pylori is a useful method to detect clarithromycin-resistant strains of H. pylori easily.