应用变性高效液相色谱法检测幽门螺杆菌对克拉霉素的耐药性

Detection of clarithromycin resistance in Helicobacter pylori by denaturing high performance liquid chromatography assay

目的建立DHPLC快速检测Hp对克拉霉素耐药性的新方法。方法抽提56份经纸片琼脂扩散法鉴定为克拉霉素耐药的Hp菌株DNA及其对应的胃粘膜组织标本DNA,通过PCR技术扩增23SrRNA区187bp基因,运用DHPLC技术对PCR产物进行突变分析,并进行DNA测序。结果 56份克拉霉素耐药的Hp菌株DNA经DHPLC分析,有51份存在异源双链峰,测序证实均存在点突变,5份无异源双链峰,测序证实无点突变,故DHPLC对基因突变耐药菌株的敏感性和特异性为100%,而且56份组织标本DNA检测结果与菌株DNA检测结果一致。结论 DHPLC可应用于Hp对克拉霉素耐药性的快速检测,具有广阔的临床应用前景。

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.