PCR-RFLP结合克隆测序监测乙肝病毒拉米夫定耐药突变株的产生

目的 建立PCR结合酶切的方法监测慢性乙型肝炎患者体内拉米夫定耐药突变株的产生，并与PCR产物克隆后测序相结合。了解此方法的可靠性和可行性，同时用此方法筛查50例应用拉米夫定治疗的慢性乙型肝炎患者中耐药株的发生情况。方法 拉米夫定治疗的慢性乙型肝炎患者50例，治疗时间9个月-24个月，设计错配PCR结合限制性片段长度多态性方法，快速检测患者体内乙型肝炎病毒（HBV）酪氨酸-蛋氨酸-天门冬氨酸-天门冬氨酸（Tyrosine-Methionine-Aspartic acid-Aspartic acid,YMDD)变异株的发生情况，对筛检耐药株阳性的标本应用PCR产物克隆后测序加以证实。结果 在50例服用拉米夫定患者中发现9斧正患者在用药超过9个月时出现拉米夫定耐药突变株（YMDD变异株），其中YIDD变异5例，YVDD变异4例，后者有3例合并有1526M突变。结论 本方法在检测YMDD变异方面具人快速简便的特点，经克隆后测序证实，具有较好的可靠性。

Detection of HBV lamivudine-resistant mutants with the method of PCR-RFLP in combination with PCR clone sequencing

Objective To monitor the lamivudine resistant HBV mutants in patients treated with lamivudine. Methods Using a pair of mismatched primers, a novel method of PCR RFLP in combination with clone sequencing was designed and HBV YMDD mutant was analysed in 50 chronic hepatitits B patients treated with lamivudine. Results Nine patients were infected with YMDD mutants. The mutants were verified by sequencing. Five patients have YIDD mutants. Four patients have YVDD mutants and three of them have concomitant L526M mutants. Conclusion The novel method of PCR RFLP is easy to operate and reliable in monitoring the YMDD mutants though it cannot detect the mutations outside YMDD motif. [