应用限制性片段长度多态性检测幽门螺杆菌对克拉霉素的耐药性

Detection of resistance of clarithromycin to Helicobacter pylori by restriction fragment length polymorphism assay

目的建立限制性片段长度多态性(restriction fragment length polymorphism,RFLP)快速检测HP对克拉霉素耐药性的方法。方法抽提37例经纸片琼脂扩散法鉴定为克拉霉素耐药的HP菌株DNA及其对应的胃黏膜组织标本DNA,通过PCR技术扩增23S rRNA区425 bp基因,运用RFLP技术,采用BbsⅠ和BsaⅠ内切酶对PCR产物进行酶切分析,并进行DNA测序。结果37例克拉霉素耐药的HP菌株DNA经RFLP分析,21例能被BbsⅠ内切酶切开,13例能被BsaⅠ内切酶切开,3例不能被BbsⅠ和BsaⅠ内切酶切开,而且37例组织标本DNA检测结果与菌株DNA检测结果一致。结论RFLP可应用于HP对克拉霉素耐药性的快速检测,为临床用药提供指导。

Objective To evaluate the restriction fragment length polymorphism(RFLP)assay for the rapid detection of clarithromycin resistance to Helicobacter pylori.Methods A 425 bp fragment of the 23S rRNA gene was amplified using DNA from 37 clinical Helicobacter pylori isolates,which were resistant to clarithromycin by agar dilution,and the homologous gastric biopsies.The RFLP for the detection of the mutations digested by BbsⅠand BsaⅠenzyme,was carried out on all strains and biopsies.Sequencing was performed on the all PCR products.Results Of the 37 resistant isolates,21 were digested with BbsⅠ,13 were digested with BsaⅠand 3 were not digested with BbsⅠor BsaⅠ.Sequencing revealed no point mutations in all the 3 resistant isolates.Furthermore,there was the same results from the homologous gastric biopsies.Conclusion RFLP assay is a rapid and valid tool for rapid assessment of clarithromycin resistance to Helicobacter pylori,and may have extensive perspective of clinical application.