Applied Molecular Virology Laboratory, Institut Pasteur Korea, 696 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do 13488, Korea.
Catholic University Liver Research Center, The Catholic University of Korea, Seoul 06591, Korea.
Viruses. 2020 Aug 24;12(9):928. doi: 10.3390/v12090928.
Hepatitis B virus (HBV) is a para-retrovirus that reverse transcribes its pregenomic RNA into relaxed circular DNA inside viral nucleocapsids. The number of HBV genomes produced in vitro is typically quantified using commercial silica-membrane-based nucleic acid purification kits to isolate total DNA followed by HBV-specific quantitative PCR (qPCR). However, despite the convenience of commercial kits, this procedure is costly and time-consuming due to multiple centrifugation steps, which produce unnecessary waste. Here, we report a rapid, cost-effective, and environmentally friendly total DNA preparation method. The assay is based on the simple incubation of detergent and proteinase K with cells or cell-free supernatants to permeabilize cells and disrupt viral particles. After heat inactivation and subsequent centrifugation to clear the lysates, DNA samples are directly subjected to qPCR to quantify HBV genomes. As a proof of concept, the assay was developed in 12-well plates to assess intra- and extracellular HBV genome equivalents (GEqs) of stably viral-replicating cell lines (e.g., HepAD38) and HBV-infected HepG2-NTCP cells, both treated with lamivudine (LMV), an HBV replication inhibitor. Viral DNA was also prepared from the serum of patients chronically infected with HBV. To validate the assay, a representative commercial DNA isolation kit was used side-by-side to isolate intra- and extracellular HBV DNA. Both methods yielded comparable amounts of HBV GEqs with comparable LMV 50% efficient concentration (EC) values. The assay was subsequently adapted to 96- and 384-well microtiter plates using HepAD38 cells. The EC values were comparable to those obtained in 12-well plates. In addition, the calculated coefficient of variation, Z' values, and assay window demonstrated high reproducibility and quality. We devised a novel, robust, reproducible, high-throughput microtiter plate DNA preparation method suitable for quantifying HBV GEqs by qPCR analysis. This strategy enables rapid and convenient quantitative analysis of multiple viral DNA samples in parallel to investigate intracellular HBV replication and the secretion of DNA-containing viral particles.
乙型肝炎病毒 (HBV) 是一种拟逆转录病毒，它将前基因组 RNA 逆转录到病毒核衣壳内的松弛环状 DNA 中。在体外，HBV 基因组的数量通常通过商业的基于硅质膜的核酸纯化试剂盒来定量，该试剂盒用于分离总 DNA，然后进行 HBV 特异性定量 PCR (qPCR)。然而，尽管商业试剂盒很方便，但由于需要多次离心步骤，该过程既昂贵又耗时，还会产生不必要的浪费。在这里，我们报告了一种快速、经济高效且环保的总 DNA 制备方法。该测定法基于简单地孵育去污剂和蛋白酶 K 与细胞或无细胞上清液，以通透细胞并破坏病毒颗粒。热灭活后，通过离心清除裂解物，然后直接将 DNA 样品进行 qPCR 以定量 HBV 基因组。作为概念验证，该测定法在 12 孔板中进行开发，以评估稳定复制病毒的细胞系（例如 HepAD38）的细胞内和细胞外 HBV 基因组当量 (GEq)，以及用拉米夫定 (LMV) 处理的 HBV 感染 HepG2-NTCP 细胞，HBV 复制抑制剂。病毒 DNA 也从慢性 HBV 感染患者的血清中制备。为了验证该测定法，使用了代表性的商业 DNA 分离试剂盒来分离细胞内和细胞外 HBV DNA。两种方法都产生了可比较数量的 HBV GEq，且拉米夫定 50%有效浓度 (EC) 值相当。该测定法随后被用于 HepAD38 细胞的 96 孔和 384 孔微滴定板。EC 值与在 12 孔板中获得的值相当。此外，计算的变异系数、Z' 值和测定窗口表明了高重现性和质量。我们设计了一种新颖、稳健、可重复的高通量微滴定板 DNA 制备方法，适用于通过 qPCR 分析定量 HBV GEq。该策略能够快速方便地对多个病毒 DNA 样本进行平行定量分析，以研究细胞内 HBV 复制和含 DNA 病毒颗粒的分泌。
