Oligonucleotide microarray: A new rapid method for screening the 23S rRNA gene of Helicobacter pylori for single nucleotide polymorphisms associated with clarithromycin resistance

Abstract

Resistance to antibiotics in Helicobacter pylori is increasing and becoming a serious problem in eradication treatment. Resistance of H. pylori to clarithromycin has been found to be associated with 2142 A-to-G, 2143 A-to-G and 2182 C-to-T point mutations in the 23S rRNA gene. Thus, the purpose of the present study was to develop a new method to analyze single nucleotide polymorphism (SNPs) of 23S rRNA gene using oligonucleotide microarray and to determine the prevalence of each mutation in H. pylori-positive patients. Gastric tissue biopsy specimens were obtained from patients undergoing upper gastrointestinal endoscopy. After DNA extraction, asymmetric PCR was employed to prepare single-stranded target DNA labeled with a fluorescent dye. The PCR products that amplified a portion of 23S rRNA from H. pylori isolates were hybridized on DNA microarray, specific for SNP genotyping and mutation detection. The optimal signal intensity and efficiency of hybridization were observed for capture probes in the detection of DNA sequence variation. The relevant mutation was confirmed by DNA sequencing analysis. Fifty-four gastric biopsy specimens yielded H. pylori-positive results and were studied to detect mutations in the 23S rRNA gene. There were no samples with A-to-G transition at position 2142. The 2143 A-to-G and 2182 C-to-T mutations were present in 11.11% and 12.96% of H. pylori strains examined, respectively. The relevant mutation was confirmed by analysis of DNA sequencing to be the same as that described at position 2142, 2143 and 2182 using oligonucleotide microarray. Oligonucleotide microarray of the PCR product permits a rapid and accurate screening of SNPs of 23S rRNA gene from H. pylori. It is now possible to apply this hybridization technology in clinical diagnosis and treatment.