High-resolution melting curve analysis of levofloxacin-resistant gyrA gene mutation in Helicobacter pylori

Abstract

Objective To optimize and establish a PCR-high resolution melting curve (HRM) for analysis of levofloxacin-resistant gyrA gene mutation in Helicobacter pylori, and to provide evidence for clinical selection of individualized Helicobacter pylori eradication regimens. Methods Gastric mucosal tissues were collected from 45 patients with peptic ulcer who underwent gastroendoscopy at the Department of Gastroenterology, Dongtai Hospital Affiliated to Nantong University between May 2013 and October 2013. After culture under microaerobic condition (5% O2, 10% CO2, 85% N2) , 23 clinical isolates of Helicobacter pylori strains were obtained and subjected to levofloxacin susceptibility test by E-test. Total DNA was extracted from the gastric mucosa and these clinical isolates. Partial fragments of gyrA gene were amplified by PCR and then sequence analysis was performed. GyrA gene mutations were analyzed by PCR-HRM. Results The rate of levofloxacin resistance was 47.8% (11/23) . GyrA gene was mutated in 10 strains of the drug-resistant strains, presenting chiefly as C261A, G271T and A272G+ G514A. There were no mutations of the gyrA gene in 9.1% (1/11) of the levofloxacin resistant strains and all sensitive strains. The specificity and reproducibility of the PCR-HRM method was 100%, with a minimum detection limit of 102 copies/μl. Conclusion The PCR-HRM method may accurately and rapidly detect the gyrA gene mutation in Helicobacter pylori. Key words: Helicobacter pylori; Drug resistance; Levofloxacin; Gene; Mutation; High resolution melting curve