HBV耐拉米夫定多聚酶基因变异检测方法的建立  

Establishment of a method for detection of lamivudine-resistant mutants in sequence of HBV polymerase gene

作　　者：张伟三[1] 丁静娟[1] 门晓燕[1] 

机构地区：[1]贵阳医学院附属医院,550004

出　　处：《现代实用医学》2002年第7期346-348,共3页Modern Practical Medicine

摘　　要：目的　建立一种简便、准确、实用的乙型肝炎病毒 (HBV)耐拉米夫定多聚酶基因变异检测方法。　方法　根据已公布的HBVDNA序列 ,在HBV多聚酶基因区设计并合成了两对寡核苷酸引物 ,其中一只为错配引物 ;应用巢式错配聚合酶链反应 (PCR)特异性扩增含有HBV多聚酶基因YMDD基序的片段 ,扩增产物用限制性内切酶 (NdeⅠ )酶切 ,琼脂糖凝胶电泳 ,观察酶切后目的片段的限制性片段长度多态性 (RFLP)图谱。部分标本进行DNA测序。　结果　 (1)所建立的巢式错配PCR RFLP法 ,从DNA提取到酶切后电泳分析仅需要 11小时 ;灵敏度高 ,可以检测到 10 3 copies/ml的HBVDNA ;特异性强 ,结果准确 ,经DNA测序证实。 (2 )检测 2 0例长期服用拉米夫定的慢性乙型肝炎患者的系列血清 ,发现YMDD变异者 (45 % ) 9例 ,YMDD野毒株者 (5 5 % ) 11例 ,表明该法可有效地鉴别HBVYMDD野毒株与变异株。　结论　本实验所建立的用于检测HBV耐拉米夫定多聚酶基因变异的巢式错配PCR RFLP方法简单、敏感、特异 ,适用于一般实验室及大规模的人群调查。Objective To establish a simple,accurate and practical method for detection of lamivudine resistant mutants in sequence of polymerase gene of hepatitis B virus(HBV). Methods Two pair of primers of oligonucleotide were synthesized to amplify approximately 119bp of HBV polymerase gene spanning YMDD motif,where mutations associated with resistance to lamivudine invariably occur. One of which was misparing primer. Then HBV polymerase gene YMDD motif were amplified by nested mismatched polymerase chain reaction (PCR). The PCR products were digested with NdeⅠand subjected to electrophoresis on agarose gels.The patterns of restriction fragment length polymorphism (RFLP) of HBV polymerase gene were distinguished. Results The method was extremely rapid, sensitive, specific and accurate. Serial serum of 20 patients with chronic hepatitis B following long time of lamivudine therapy were detected by this method, 11 patients(55%) had not found mutations,9 patients (45%) developed mutations in the YMDD motif of HBV polymerase gene. It was shown that HBV polymerase gene mutations could be confirmed .The result were confirmed by DNA sequencing. Conclusion The nested mismatched PCR RFLP method is a rapid,simple,specific and sensitive technique for detection of lamivudine resistance associated mutations in sequence of HBV polymerase gene.This method is suitable for routine laboratories and large scale population studies.

关 键 词：HBVJ 拉米夫定 多聚酶基因 耐药性 基因变异 巢式错配PCR-RELP 

分 类 号：R373.21[医药卫生—病原生物学]