高通量测序检测拉米夫定抗乙肝病毒治疗的耐药突变

Determination of Lamivudine-resistant mutation against hepatitis B virus via high-throughput sequencing

摘要:

目的：建立一种高通量、快速检测拉夫米定耐药突变的新方法，指导临床抗病毒治疗药物的选择。方法：选取 150例慢性乙型肝炎患者血清标本为研究对象，乙型肝炎病毒（Hepatitis B virus，HBV）DNA定量结果均阳性（拷贝数≥１03拷贝/ml），设计针对聚合酶P区的扩增引物，经 PCR扩增及磁珠分离，制备焦磷酸测序单链模板，并在 PyroMark ID遗传分析系统上进行焦磷酸测序，检测拉夫米定耐药突变位点。结果：PCR扩增得到 HBV P区产物，焦磷酸测序检测突变频率具有极高的灵敏性、特异性、稳定性的高通量的特征且与拉夫米定用药时间存在相关性。用药时间越长，突变率越高，易出现耐药。结论：新型、高通量焦磷酸测序法用于拉米夫定耐药突变检测，能满足临床批量用药检测及治疗方案设计的需要。

Abstract:

Objective：To establish a high-throughput method which can rapidly detect Lamivudine-resistant mutation against hepati－tis B virus（HBV） to guide clinical medication. Methods：Serum samples of 150 patients with chronic hepatitis B（CHB），whose quan－titative HBV DNA results were positive（copy number ≥103），were determined by pyrosequencing for detecting Lamivudine-resistantly mutational site via PyroMark ID genetic analysis systems. In order to prepare single-stranded template of pyrosequencing，samples were amplified by nested PCR and were separated by magnetic bead and amplification primer of the polymerase in P region was designed. Results：HBV P region products were attainted by amplified PCR and mutation frequencies of pyrosequencing were characterized by high sensitivity，specificity，stability and were in correlation with Lamivudine-treatment time. With the increase of Lamivudine treatment time，mutation frequencies and drug resistance were increased. Conclusions：High-throughput pyrosequencing may meet the needs of clinical drug testing and design of treatment protocols，which can detect Lamivudine-resistant mutation of HBV in patients treated with Lamivudine．