A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes
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 DN...
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doaj-d2b02a55d0c944acb2416b1a07d407992020-11-25T03:43:34ZengMDPI AGViruses1999-49152020-08-011292892810.3390/v12090928A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus GenomesEunji Jo0Jaewon Yang1Alexander Koenig2Seung Kew Yoon3Marc P. Windisch4Applied Molecular Virology Laboratory, Institut Pasteur Korea, 696 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do 13488, KoreaApplied Molecular Virology Laboratory, Institut Pasteur Korea, 696 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do 13488, KoreaApplied Molecular Virology Laboratory, Institut Pasteur Korea, 696 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do 13488, KoreaCatholic University Liver Research Center, The Catholic University of Korea, Seoul 06591, KoreaApplied Molecular Virology Laboratory, Institut Pasteur Korea, 696 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do 13488, KoreaHepatitis 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<sub>50</sub>) values. The assay was subsequently adapted to 96- and 384-well microtiter plates using HepAD38 cells. The EC<sub>50</sub> 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.https://www.mdpi.com/1999-4915/12/9/928hepatitis B virusviral DNAgenome equivalentsintracellularextracellularDNA preparation |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Eunji Jo Jaewon Yang Alexander Koenig Seung Kew Yoon Marc P. Windisch |
spellingShingle |
Eunji Jo Jaewon Yang Alexander Koenig Seung Kew Yoon Marc P. Windisch A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes Viruses hepatitis B virus viral DNA genome equivalents intracellular extracellular DNA preparation |
author_facet |
Eunji Jo Jaewon Yang Alexander Koenig Seung Kew Yoon Marc P. Windisch |
author_sort |
Eunji Jo |
title |
A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes |
title_short |
A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes |
title_full |
A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes |
title_fullStr |
A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes |
title_full_unstemmed |
A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes |
title_sort |
simple and cost-effective dna preparation method suitable for high-throughput pcr quantification of hepatitis b virus genomes |
publisher |
MDPI AG |
series |
Viruses |
issn |
1999-4915 |
publishDate |
2020-08-01 |
description |
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<sub>50</sub>) values. The assay was subsequently adapted to 96- and 384-well microtiter plates using HepAD38 cells. The EC<sub>50</sub> 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. |
topic |
hepatitis B virus viral DNA genome equivalents intracellular extracellular DNA preparation |
url |
https://www.mdpi.com/1999-4915/12/9/928 |
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