Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure.
Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure.
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DOI：
10.1136/gutjnl-2022-328380
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发表时间：
2023-05
期刊：
影响因子：
24.5
通讯作者：
Dandri, Maura
中科院分区：
文献类型：
--
作者：
Allweiss, Lena;Testoni, Barbara;Yu, Mei;Lucifora, Julie;Ko, Chunkyu;Qu, Bingqian;Luetgehetmann, Marc;Guo, Haitao;Urban, Stephan;Fletcher, Simon P.;Protzer, Ulrike;Levrero, Massimo;Zoulim, Fabien;Dandri, Maura
乙型肝炎病毒 (HBV) 治疗的主要目标是减少或灭活肝内病毒共价闭合环状 DNA (cccDNA)。因此，精确的 cccDNA 定量在临床前和临床研究中至关重要。 Southern blot (SB) 允许 cccDNA 可视化，但缺乏灵敏度且非常费力。定量 PCR (qPCR) 没有此类限制，但由于病毒复制中间体 (RI) 的共检测，可能会出现定量不准确的情况。不同样品、保存条件、DNA 提取、核酸酶消化方法和 qPCR 策略的使用阻碍了标准化。在 ICE-HBV 联盟内，在六个实验室对肝组织和细胞培养物中 cccDNA 分离和 qPCR 定量的可用和新颖方案进行了比较，以制定最佳实践的循证指南。交换参考材料（感染 HBV 的人源化小鼠肝脏和 HepG2-NTCP 细胞）以进行交叉验证。每组比较不同的 DNA 提取方法（Hirt 提取、有或没有蛋白酶 K 处理的总 DNA 提取 (+PK/−PK)）和核酸酶消化方案（质粒安全的 ATP 依赖性 DNase (PSD)、T5 核酸外切酶、核酸外切酶 I/III）。通过 qPCR 和 SB 分析样品。 Hirt 和−PK 提取减少了共存的 RI 形式。然而，qPCR 检测到了 cccDNA 和无蛋白的松弛环状 HBV DNA (pf-rcDNA) 形式。 T5 和 Exo I/III 核酸酶可有效去除所有 RI 形式。相比之下，PSD不消化pf-rcDNA，但不太容易引起cccDNA过度消化。在稳定的组织（例如 Allprotect）中，核酸酶对 cccDNA 具有不利影响。我们在此提出一个全面的基于证据的指南，用于使用可用的 qPCR 检测来优化、控制和验证 cccDNA 测量。
A major goal of curative hepatitis B virus (HBV) treatments is the reduction or inactivation of intrahepatic viral covalently closed circular DNA (cccDNA). Hence, precise cccDNA quantification is essential in preclinical and clinical studies. Southern blot (SB) permits cccDNA visualisation but lacks sensitivity and is very laborious. Quantitative PCR (qPCR) has no such limitations but inaccurate quantification due to codetection of viral replicative intermediates (RI) can occur. The use of different samples, preservation conditions, DNA extraction, nuclease digestion methods and qPCR strategies has hindered standardisation. Within the ICE-HBV consortium, available and novel protocols for cccDNA isolation and qPCR quantification in liver tissues and cell cultures were compared in six laboratories to develop evidence-based guidance for best practices. Reference material (HBV-infected humanised mouse livers and HepG2-NTCP cells) was exchanged for cross-validation. Each group compared different DNA extraction methods (Hirt extraction, total DNA extraction with or without proteinase K treatment (+PK/−PK)) and nuclease digestion protocols (plasmid-safe ATP-dependent DNase (PSD), T5 exonuclease, exonucleases I/III). Samples were analysed by qPCR and SB. Hirt and −PK extraction reduced coexisting RI forms. However, both cccDNA and the protein-free relaxed circular HBV DNA (pf-rcDNA) form were detected by qPCR. T5 and Exo I/III nucleases efficiently removed all RI forms. In contrast, PSD did not digest pf-rcDNA, but was less prone to induce cccDNA overdigestion. In stabilised tissues (eg, Allprotect), nucleases had detrimental effects on cccDNA. We present here a comprehensive evidence-based guidance for optimising, controlling and validating cccDNA measurements using available qPCR assays.
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DOI：
10.1007/978-1-62703-484-5_13
发表时间：
2013
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
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通讯作者：
Guo, Haitao
影响因子：
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Zoulim, F
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DOI：
10.3390/v14030557
发表时间：
2022-03-08
期刊：
Viruses
影响因子：
--
作者：
Bustamante-Jaramillo LF;Fingal J;Blondot ML;Rydell GE;Kann M
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5.4
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Zhang, YY;Summers, J
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Long Q;Yan R;Hu J;Cai D;Mitra B;Kim ES;Marchetti A;Zhang H;Wang S;Liu Y;Huang A;Guo H
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Guo H