Quantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in the liver of HBV-infected patients by LightCycler™ real-time PCR
Quantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in the liver of HBV-infected patients by LightCycler™ real-time PCR
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DOI：
10.1016/j.jviromet.2004.02.006
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发表时间：
2004-06-15
影响因子：
3.1
通讯作者：
Sacks, SL
中科院分区：
文献类型：
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作者：
Singh, M;Dicaire, A;Sacks, SL
乙型肝炎病毒 (HBV) 抗病毒药物可降低病毒载量并改善肝脏组织学，但其对共价闭合环状 DNA (cccDNA)（HBV 转录模板）的影响尚未得到广泛研究。本研究评估了新设计的基于 LightCycler(TM) 的定量 cccDNA PCR 测定。使用 HBV cccDNA 特异性引物建立了 2.5 x 10(1) 至 1 x 10(9) 拷贝/测定的线性范围，并使用总 HBV DNA (tDNA) 特异性引物建立了 2.5 x 10(1) 至 2 x 10(9) 拷贝/测定的线性范围。 β-珠蛋白用于估计每个 PCR 反应中的细胞数量。使用 ATP 依赖性 DNase 进行酶消化，将 cccDNA:RC DNA（松弛环状 DNA）的分析特异性提高至大于 1:10,000。在盲法条件下，对 6 名患者（3 名 HBV 感染者和 3 名未感染者）的 1 mg 肝活检中提取的 DNA 进行了分析；其中 3 例 cccDNA 和 tDNA 呈阳性，3 例呈阴性。在接受拉米夫定治疗的 HBV 感染患者的移植前活检中，检测到大约 6 x 10(3) 个 cccDNA 拷贝/mg 组织。移植后连续肝活检的 HBV cccDNA 和 tDNA 均为阴性。一名未接受过抗病毒治疗的 HBV 感染肝硬化患者的 cccDNA 拷贝数为 3.7 x 10(4)/mg 肝组织。另一名具有高 HBV 病毒载量史的初治患者每毫克组织有 1 x 10(5) cccDNA 拷贝（每毫克组织有 4 x 10(6) tDNA 拷贝）。进一步的研究是有必要的，但这种采用 LightCycler(TM) 系统的新型检测方法所提供的高水平敏感性、特异性、快速性和准确性表明，它可用于监测抗病毒治疗。 (C) 2004 Elsevier B.V. 保留所有权利。
Antivirals for hepatitis B virus (HBV) reduce viral load and improve liver histology, however, their effect on covalently closed circular DNA (cccDNA), the HBV transcriptional template, has not been extensively examined. This study evaluated a newly designed LightCycler(TM)-based quantitative cccDNA PCR assay. A linear range of 2.5 x 10(1) to 1 x 10(9) copies/assay using primers specific for HBV cccDNA and 2.5 x 10(1) to 2 x 10(9) copies/assay using primers specific for total HBV DNA (tDNA) was established. beta-Globin was used to estimate the number of cells in each PCR reaction. Enzymatic digestion with an ATP-dependent DNase improved the analytic specificity to a greater than 1: 10,000 ratio of cccDNA:RC DNA (relaxed circular DNA). One-tenth of the extracted DNA from 1 mg of liver biopsy, was analyzed from six patients, three HBV-infected and three uninfected individuals, under blinded conditions; three were found positive and three negative for cccDNA and tDNA. Approximately 6 x 10(3) copies of cccDNA/mg of tissue were detected in a pre-transplant biopsy from an HBV-infected patient treated with lamivudine. Sequential post-transplant liver biopsies were negative for both HBV cccDNA and tDNA. An HBV-infected patient with cirrhosis who was antiviral therapy naive had 3.7 x 10(4) copies of cccDNA/mg of liver tissue. Another treatment-naive patient with a history of high HBV viral load had 1 x 10(5) copies of cccDNA/mg of tissue (4 x 10(6) copies of tDNA/mg of tissue). Further studies are warranted but the high level of sensitivity, specificity, rapidity and accuracy provided by this novel assay with the LightCycler(TM) system indicate that it could be useful for monitoring antiviral therapy. (C) 2004 Elsevier B.V. All rights reserved.