限制性片段长度多态性分析拉米夫定治疗引起乙型肝炎病毒耐药基因变异的研究

Detection of the mutation in HBV polymerase gene by RFLP PCR method in hepatitis B patients treated with lamivudine

目的 应用聚合酶链反应限制性片段长度多态性分析方法 (PCRRFLP) ,研究拉米夫定治疗引起慢性乙型肝炎患者血清中乙型肝炎病毒耐药基因 (HBVDNA)变异。方法 收集 2 4 0例用拉米夫定治疗 5 2～ 78周慢性乙型肝炎患者的血清标本 ,应用PCRRFLP方法扩增HBV 5 5 2 ,5 2 8两个基因位点片段 ,用限制性内切酶NdeⅠ ,NlaⅢ酶切分析 ,同时测定HBVDNA含量 ,并用DNA序列分析测定 3例患者HBVDNAP区基因序列 (1例野毒株 ,1例M5 5 2V伴L5 2 8M变异 ,1例M5 5 2I变异 )。结果 2 4 0例患者应用拉米夫定治疗 5 2～ 78周后 ,5 1例血清HBVDNA出现YMDD变异 ,其中M5 5 2V变异 38例 ,M5 5 2I变异 13例 ,L5 2 8M和M5 5 2V同时变异 2 6例 ,L5 2 8M和M5 5 2I同时变异 1例。与定量PCR比较 ,可以测定YMDD变异的最低含量为 1× 10 4 copies mlHBVDNA序列分析结果与RLFP测定一致。结论 PCRRLFP方法是一种快速、简便的检测HBVDNA聚合酶变异的方法 ,可以用来筛查大量的血清标本 。

Objective To investigate the mutation of HBV polymerase gene in chronic hepatitis B patients treated with lamivudine Methods The restriction fragment length polymorphism(RFLP)assay for HBV DNA sequence determination at the codon 528 and 552 in the HBV polymerase gene associated with lamivudine resistance in vitro HBV DNA samples extracted from sera of 240 patients were subjected to PCR amplification with primer pairs F2/R2(552), F3/R2(528) Each PCR product was digested with Nde Ⅰ or Nla Ⅲ Results Serum HBV DNA mutation was found in 51/240 patients (38/51M552V、26/38L528M、13/51M552I) after therapy for 52 weeks. DNA sequence analysis was performed on samples of 3 patients, and the results were consistent with those of RFLP assay.Conclusion The RFLP assay was able to detect the mutation of HBV DNA at codon 552 and 528 which are the principal site of HBV DNA resistant to lamivudine The specific PCR method for HBV DNA mutation is rapid, simple and specific