CAJ | 학술논문

目的：建立基于滚环复制的、高效扩增血清松弛环状乙型肝炎病毒DNA（HBV RC-DNA）全长基因组的方法。方法以60例慢性乙型肝炎患者血清HBV RC-DNA为研究对象，利用T4 DNA polymerase和T4 DNA ligase，封闭HBV RC-DNA基因组上的缺口，使之成为闭合环状结构；再通过Phi29 DNA polymerase的作用，对闭合环状的HBV基因组进行滚环复制，以滚环复制后的RC-DNA为模板扩增HBV全长基因组。结果成功建立了基于滚环复制的HBV RC-DNA全长基因组扩增方法，可从病毒载量为108~104拷贝/ml样本中扩增出HBV全长基因组。对于107~105拷贝/ml血清样本来说，使用基于RC-DNA滚环复制的全长基因组扩增方法，与以往的一步法扩增相比，扩增效率显著提高。结论基于滚环复制的HBV RC-DNA全长基因组扩增方法具有较高的灵敏度。该方法的建立为H BV全基因组研究提供了一种新的、有效的工具，有望在基础和临床研究中广泛应用。
목적：건립기우곤배복제적、고효확증혈청송이배상을형간염병독DNA（HBV RC-DNA）전장기인조적방법。방법이60례만성을형간염환자혈청HBV RC-DNA위연구대상，이용T4 DNA polymerase화T4 DNA ligase，봉폐HBV RC-DNA기인조상적결구，사지성위폐합배상결구；재통과Phi29 DNA polymerase적작용，대폐합배상적HBV기인조진행곤배복제，이곤배복제후적RC-DNA위모판확증HBV전장기인조。결과성공건립료기우곤배복제적HBV RC-DNA전장기인조확증방법，가종병독재량위108~104고패/ml양본중확증출HBV전장기인조。대우107~105고패/ml혈청양본래설，사용기우RC-DNA곤배복제적전장기인조확증방법，여이왕적일보법확증상비，확증효솔현저제고。결론기우곤배복제적HBV RC-DNA전장기인조확증방법구유교고적령민도。해방법적건립위H BV전기인조연구제공료일충신적、유효적공구，유망재기출화림상연구중엄범응용。
ObjectiveTo establish an efficient method for amplification of full-length HBV genome with relaxed-circular serum DNA (RC-DNA).MethodsHBV RC-DNA obtained from 60 patients with chronic hepatitis B was involved in this study. First, T4 DNA polymerase and T4 DNA ligase were used to circularize the genome of HBV RC-DNA so that it became a closed circular form. Second, Phi29 DNA polymerase was applied for the rolling circle replication of closed circular RC-DNA, the product of which was then used as the template for amplification of fulllength HBV genome.ResultsWe successfully established the method for amplification of full-length HBV genome with RC-DNA, which was capable to amplify the complete HBV genome from serum samples with viral load ranging between108 to 104 copies/ml. Compared with the one-step amplification method described previously, our method was more efficient for serum samples with viral load varied from 107 to 105 copies/ml.ConclusionThe method for amplification of full-length HBV genome based on rolling circle replication of RC-DNA was efficient, and it might be a new and powerful tool for the basic and clinical study of HBV genome.