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目的采用新型淬灭剂结合荧光PCR检测幽门螺杆菌克拉霉素耐药基因突变的检测方法。方法根据幽门螺杆菌23S r DNA第2142和2143两个基因多态性位点设计引物和标记新型荧光淬灭剂的探针;提取幽门螺杆菌菌体总DNA,采用实时荧光定量PCR方法对收集的55株幽门螺杆菌临床分离株进行检测,同时与Taq Man探针和直接测序方法结果进行比对,进一步评价其临床实用性。结果本试验建立的方法能特异性地甄别23S r DNA第2142和2143位点的基因多态性,除对应位点的探针出现荧光信号外,其他探针均为阴性。该方法的变异系数小于5%,样本灵敏度可达到1 copy/反应。对收集的55株幽门螺杆菌临床分离株进行检测,检测到AA型菌株36株(65.45%)、AG型菌株15株(27.27%)和GA型菌株4株(7.27%),与Taq Man探针及直接测序结果均一致。结论本试验所建立的方法能有效甄别幽门螺杆菌克拉霉素耐药基因突变位点A2142G和A2143G。
Objective To detect the mutation of clarithromycin resistance gene of Helicobacter pylori by a novel quencher combined with fluorescent PCR. Methods Based on the polymorphisms of 2142 and 2143 of Helicobacter pylori (23S rDNA), primers were designed and labeled with a new fluorescent quencher. The total DNA of Helicobacter pylori was extracted and quantified by real-time fluorescence quantitative PCR Of 55 H. pylori clinical isolates were tested and compared with Taq Man probe and direct sequencing method results to further evaluate its clinical utility. Results The method established in this study could specifically identify the polymorphisms of the 2142nd 2143rd of 23S rDNA. All the probes were negative except for the fluorescence signals of the corresponding sites. The method of variation coefficient of less than 5%, the sample sensitivity can be achieved 1 copy / reaction. A total of 55 clinical isolates of Helicobacter pylori were collected and detected 36 strains (65.45%) of AA strains, 15 strains (27.27%) of AG strains and 4 strains (7.27%) of GA strains, Needle and direct sequencing results are the same. Conclusion The method established in this study can effectively identify the A2142G and A2143G mutation sites of Clarithromycin resistance in Helicobacter pylori.