PCR-RFLP检测拉米夫定抗HBV感染中聚合酶YMDD变异  

Analysis of YMDD mutants in hepatitis B virus infected patients with lamivudine therapy by restriction fragment length polymorphism (RFLP) of the PCR products

作　　者：杨丹红[1] 梁伟峰[1] 赵年丰[1] 范骏[1] 谢远军[2] 潘小平[1] 陈智[1] 

机构地区：[1]浙江大学医学院附属第一医院传染科 [2]浙江中医学院针推系

出　　处：《中华微生物学和免疫学杂志》2002年第4期361-364,共4页Chinese Journal of Microbiology and Immunology

基　　金：浙江省卫生厅科研基金资助项目 ( 2 0 0 0A0 96 )

摘　　要：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 analyze the emergence of YMDD variants in patients with hepatitis B treated with lamivudine. Methods The aminoacid substitution from methionine or isoleucine at the YMDD(tyrosine, methionine, aspartate, aspartate) motif at the HBV polymerase gene is main mutation responsible for resistant to lamivudine treatment. Generated from a fragment of domain C of the polymerase gene from clinical samples that patients' HBV DNA became positive again and they were negative even during lamivudine therapy. Variants were detected by cleavage of the products of the three PCRs with following enzymes: FokⅠ, SspⅠ, Alw441. The results of PCR-RFLP were analysed by 8.4% polypropylene acidemide gel electrophoresis. The PCR-RFLP assay were compared to direct sequencing. Results There were 33 patients' HBV DNA became positive again and two patients' HBV DNA have been positive for one year. The YMDD variants were detected in 14 of 35 serum samples with 4 cases YIDD variant 6 cases YVDD variant and 1 case YI/MDD in concordance with direct sequening. The other 3 patients' samples showed YI/VDD mutations, which compare to the direct sequence and were concordant with direct sequening too. The PCR-RFLP assay of the mixed sera of YIDD and YVDD variants are the same as that of the serum of YI/VDD variant. Conclusion The PCR-RFLP is suitable for fast detection of YMDD variants of viral polymerase in hepatitis B patients with lamivudine therapy.

关 键 词：聚合酶YMDD YMDD变异 限制性片段长度多态性 乙型肝炎病毒 拉米夫定 乙型肝炎 聚合酶链反应 

分 类 号：R346[医药卫生—基础医学]