一种高效扩增血清松弛环状乙型肝炎病毒DNA全长基因组方法的研究

Study on an efficient method for amplification of full-length HBV genome with relaxed-circular serum DNA

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HBV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。

Objective To establish an efficient method for amplification of full-length HBV genome with relaxed-circular serum DNA（RC-DNA）.Methods HBV 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,Results We 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 between10~8 to 10~4 copies/ml.Compared with the one-step amplification method described previously,our method was more efficient for serum samples with viral load varied from 10~7 to 10~5 copies/ml.Conclusion The 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.