Detection of clarithromycin resistance in Helicobacter pylori by denaturing high performance liquid chromatography assay
WU Wen-bing1, CAI Peng-wei2, FANG Chao-ying3, SHEN Jing1, CHEN Wen1
1.Department of Laboratory Medicine, Fujian Provincial Hospital,Provincial Clinical College of Fujian Medical University, Fuzhou 350001, China; 2.Department of Laboratory Medicine, Fujian Provincial South Hospital,Provincial Clinical College of Fujian Medical University, Fuzhou 350001, China; 3.Department of Endoscope Center, Fujian Provincial Hospital,Provincial Clinical College of Fujian Medical University, Fuzhou 350001, China
Abstract：The aim of this study is to evaluate the denaturing high performance liquid chromatography (DHPLC) assay for the rapid detection of clarithromycin resistance in Helicobacter pylori. A 187 bp fragment of the 23S rRNA gene was amplified using DNA from 56 clinical Helicobacter pylori isolates, which were shown to be resistant to clarithromycin by agar dilution, and directly from the homologous gastric biopsies. DHPLC and sequencing were used to detect mutations in all PCR products. For the 56 resistant isolates, 51 of the 56 resistant isolates showed heteroduplex peaks, which were easily distinguishable from the homoduplex pesks of the wild-type Helicobacter pylori reference strain. Sequencing revealed point mutations in all the 51 resistant isolates. Five of the 56 resistant isolates showed homoduplex peaks, sequencing revealed no point mutations in all the 5 resistant isolates. Our results suggested that the DHPLC assay, whose sensitivity and specificity were 100% in detecting point mutations, was a valid tool for rapid assessment of clarithromycin resistance in Helicobacter pylori and that in the future it could be used directly on biopsy specimens, avoiding the need for culture-based methods.
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2015, Vol. 31 
