Modified allele-specific primer-polymerase chain reaction method for analysis of susceptibility of Helicobacter pylori strains to clarithromycin

Abstract

Background and aim: Most clarithromycin-resistant strains of Helicobacter pylori have a mutation from adenine (A) to guanine (G) at position 2142 or 2143 of the 23S rRNA gene. Our aim in this study was to develop a polymerase chain reaction (PCR)-based assay that could determine these mutations in a single reaction tube.

Methods: We designed the forward primer FP2143G and the reverse primer RP2142G, which specifically anneal with the 2143G- and 2142G-mutated sequences, respectively, of the 23S rRNA gene of H. pylori. We also designed the forward primer FP-1 and reverse primer RP-1 upstream and downstream from the positions 2142 and 2143, respectively, to distinguish the wild-type A2142G and A2143G mutations from each other by amplicon sizes. DNA was extracted from 292 gastric tissue samples positive for rapid urease test, and the DNA underwent the PCR reaction. The results were compared with minimum inhibitory concentrations (MIC) for clarithromycin.

Results: Helicobacter pylori strains with A2142G, A2143G and wild type could be distinguished by amplicon sizes by a single PCR reaction. The genotyping results were correlated well with the MIC values for clarithromycin. The median MIC for clarithromycin of the wild-type strains was <0.015 microg/mL. Those of strains with 2142G or 2143G were > or =1.0 microg/mL.

Conclusion: Our new PCR-based assay for 23S rRNA mutations of H. pylori is a useful method for detecting clarithromycin-resistant strains of H. pylori easily.