抑制差减杂交技术筛选幽门螺杆菌对甲硝唑耐药相关DNA片段

Screening of metronidazole-resistance associated DNA fragments of Helicobacter pylori by suppression subtractive hybridization (SSH)

目的筛选幽门螺杆菌(Hp)临床分离菌株对甲硝唑耐药的相关DNA片段。方法从浙江大学医学院附属邵逸夫医院消化内科就诊患者胃粘膜活检标本中分离培养Hp。采用二倍平皿稀释法确定Hp菌株对甲硝唑的耐药性。分别选取敏感菌和耐药菌各1株,提取其基因组DNA,采用抑制差减杂交技术分别以其中1株作为被检菌,另1株作为参考菌进行基因组差异研究。结果73.8%(31/42)临床分离的Hp菌株对甲硝唑耐药。耐药株共检出10余个大小约在200～1000bp的特异性DNA片段。结论通过抑制差减杂交技术可筛选出耐药株和敏感株各自特异的DNA片段。

Objective To screen for metronidazole (MTZ)-resistance associated DNA fragments of H. pylori. Methods Forty-two clinical strains of H. pylori were isolated from gastric biopsy of patients. Sensitivity to metronidazole of H. pylori isolates was determined with two-fold dilution test. The suppression subtractive hybridization (SSH) was used to screen for the differential DNA fragments between MTZ-resistant and MTZ-sensitive strains of H. pylori. Results MTZ-resistance rate was 73.8% in 42 clinical isolates of H. pylori of this study.About 10 fragments specific to the resistant strains were obtained by SSH against sensitive strain. Conclusion MTZ-resistance specific DNA fragments and sensitive specific DNA fragments were obtained, which can be used for identifying genes associated with MTZ resistance of H. pylori.