Allele-Specific Polymerase Chain Reaction for Detection of Main gyrA Allelic Variants in Helicobacter pylori Strains

Abstract

Background: Rapid detection of resistant strains of Helicobacter pylori in human clinical samples is of major importance in clinical settings. Inability of conventional clinical laboratory techniques in detection of these strains usually leads to failure of prescribed therapeutic regimens. Objectives: The aim of this study was designing a simple and rapid allele-specific PCR (AS-PCR)-based method for detection of more frequent gyrA mutations at Asn87Lys codon, responsible for emergence of fluoroquinolone resistance in H. pylori strains. Patients and Methods: All bacterial strains were obtained from clinical biopsy samples in our laboratory. Identification of the isolates was performed by the genus- and species-specific primers and allele-specific primers, designed to match with the site of the point mutations. Samples with positive results for the designed PCR method were sequenced to verify the existence of the target mutations. Results: Point mutations in the gyrA gene at Asn87Lys codon (AAT > AAA and AAC > AAG) were detected in all standard resistant strains as well as some of clinical isolates with previously determined resistance phenotypes for fluoroquinolones. Presence of the target mutations was successfully confirmed in all the control strains by the newly designed primers and sequencing. Conclusions: The designed AS-PCR was a good and reliable method for detection of AAT > AAA and AAC > AAG point mutations in H. pylori isolates.