应用LNA-PCR法检测乙型肝炎病毒阿德福韦酯耐药位点基因突变

目的:建立简便、快速、灵敏的锁核酸(locked nucleic acid,LNA)探针实时荧光聚合酶链反应(PCR)检测方法,检测乙型肝炎病毒(hepatitis B virus,HBV)阿德福韦酯(Adefovir dipivoxil,ADV)耐药相关位点(rtA181V、rtN236T)突变。方法:通过基因测序筛选阳性样本,进而构建ADV rt181和rt236位点野生株和突变株重组质粒,设计包含扩增阿德福韦酯rtA181V和rtN236T耐药位点在内的特异性引物和LNA荧光探针,以构建的重组质粒为标准品建立实时荧光PCR反应体系,并通过与基因测序平行检测血清样本以判断检测方法的可行性与准确性。结果:所建立的LNA-PCR法能够检测102copies/ml的HBV中ADV基因突变,同时具备较高的特异性。通过对89例ADV治疗一年后HBV阳性临床样本进行检测,有8例(8.98%)rtA181V突变、5例(5.61%)rtN236T突变、2例(2.24%)rtA181V和rtN236T混合突变,检测结果与测序结果一致。结论:所建立的LNA-PCR法是一种简便、快速、灵敏的基因突变检测方法,能有效的区分单碱基突变,对慢性乙型肝炎患者德福韦治疗过程中耐药突变的监控和抗病毒药物的调整具有指导意义。

Establishment and Clinical Application of LNA-PCR Assay Detecting Hepatitis B Virus Adefovir Dipivoxil Resistance

Objective: To develop a simple, quick and sensitive method based on the LNA (locked nucleic acid) PCR for detection of rtA181V and rtN236T mutations associated with adefovir dipivoxil (ADV) resistance of hepatitis B virus (HBV). Method: Built wild strains and the mutant recombinant plasmids of ADV rt181 and rt236 by gene sequencing screening positive samples, then designed the specific primers and LNA fluorescent probes (rtA181V, rtN236T) to construct recombinant plasmid for standard real-time fluorescent PCR reaction system, and determined the feasibility and accuracy of the detection method through the parallel to gene sequencing to detect serum samples. Result: LNA-PCR assay could detect 10 ² copies/ml mutant template, with high specificity. Eighty-nine HBV DNA positive samples were from patients with ADV therapy over one year. LNA-PCR detected two (2.24%) rtA181V and rtN236T dual mutations, eight (8.98%) rtA181V mutations, five (5.61%) N236T mutations. Complete concordance between LNA-PCR and sequencing were observed with all samples. Conclusion: LNA-PCR test is a simple, fast, and sensitive method for monitoring ADV resistant mutations of HBV in patients with chronic hepatitis B.