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目的 建立用PCR 限制性片段长度多态性技术 (PCR RFLP) ,分析拉米夫定抗HBV感染中聚合酶YMDD变异方法。方法 在拉米夫定抗HBV治疗过程中HBVDNA由阴性再次转为阳性患者及HBVDNA始终保持阳性的血清达 1年或 1年以上 ,用 3对引物扩增HBVP基因的C区 ,扩增产物分别用 3个限制性内切酶分析 ,酶切结果用 8.4%聚丙烯酰胺凝胶电泳分析。检测HBVYMDD变异 ,同时血清用全自动测序仪检测YMDD变异。分析比较两者结果。结果 35例病人 ,包括 33例HBVDNA再次阳性患者 ,2例用药 1年HBVDNA未转阴。 14例病人出现YMDD变异。PCR RFLP结果为 6例YVDD变异、4例YIDD、1例YI MDD、2 1例YMDD与测序一致。另 3例PCR RFLP结果为YI VDD ,即混合变异 ,测序报告为 2例YIDD、1例YVDD ,分析相应的测序图 ,存在混合变异。并把YIDD与YVDD变异的血清混合 ,行PCR RFLP ,结果与YI VDD一致。结论 用PCR RFLP能快速、简便、灵敏地检测YMDD变异。
OBJECTIVE: To establish a polymerase chain reaction-restriction fragment length polymorphism (PCR RFLP) method for the analysis of polymerase chain reaction (YMDD) mutations in lamivudine against HBV infection. Methods In the course of anti-HBV treatment of lamivudine, HBVDNA turned from negative to positive again and HBVDNA always remained positive for more than one year or more than one year. Three pairs of primers were used to amplify the C region of HBVP gene. Three restriction endonuclease assays were performed and digestion results were analyzed by 8.4% polyacrylamide gel electrophoresis. Detection of HBVYMDD mutation, at the same time using automated sequencer serum YMDD mutation. Analysis and comparison of the two results. Results 35 patients, including 33 cases of HBVDNA again positive patients, 2 patients 1 year HBVDNA did not turn negative. 14 patients showed YMDD mutation. PCR RFLP results were 6 cases of YVDD mutation, 4 cases of YIDD, 1 case of YI MDD, 21 cases of YMDD consistent with the sequencing. The other three PCR RFLP results were YI VDD, that is, mixed variation. The sequencing report was 2 YIDD and 1 YVDD. The corresponding sequencing diagram was analyzed, and there was a mixed mutation. YIDD and YVDD mutants were mixed and PCR RFLP was performed. The result was consistent with that of YI VDD. Conclusion PCR-RFLP can detect YMDD mutation quickly, easily and sensitively.