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A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes.

ABSTRACT: 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 (EC50) values. The assay was subsequently adapted to 96- and 384-well microtiter plates using HepAD38 cells. The EC50 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.

SUBMITTER: Jo E

PROVIDER: S-EPMC7552047 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes.

Jo Eunji E Yang Jaewon J Koenig Alexander A Yoon Seung Kew SK Windisch Marc P MP

Viruses 20200824 9

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 ...[more]

PMID: 32846983

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Project description:BACKGROUND:Conventional plant DNA isolation methods are complex, time consuming and require technical expertise. These limitations were overcome using the DNA isolation kits which, however significantly add to the research costs. Hence the present study was aimed to develop a high throughput, rapid and inexpensive method of PCR ready DNA template preparation from plant materials. METHODS:Concentration of SDS in lysis buffer, amount of starting material, period and temperature for lysis were optimized for obtaining PCR ready templates from plant materials. The method was tested using RAPD and ITS specific primers for different plant species like rice, wheat, mustard, pea, soybean, pigeonpea, tomato, maize, march lilly, bougainvillea, Indian blanket flower, nerium, petunia, purple pirouette petunia, moses-in-the-cradle, golden cane palm, duranta, periwinkle, chrysanthemum and two xerophytes viz. Dipterygium glaucum and Crotaleria burhia. SSR markers RM18398 and RM26108 showed successful amplification in rice varieties Improved Pusa Basmati 1 and KS Dev 12. The effectiveness of the method was tested using fresh as well as 1 year old tissues. The storability of the lysate was also tested. RESULTS:In this report, we developed a novel method called rapid high throughput template preparation (rHTTP) method to prepare PCR ready DNA templates. Most striking feature of this technique is that it can be done anywhere where water can be boiled by any means. Using rHTTP method, PCR ready templates can be prepared in just 10 min. Robust and reproducible amplification for all the test plants were recorded with RAPD, plant ITS primers and SSR markers following this method. rHTTP methods works well for both fresh as well as old plant tissues. The lysates had a shelf life of 1 month when stored at 4 °C and 3 days when stored at room temperature. CONCLUSIONS:rHTTP method has several advantages over the other protocols like ease of execution, no requirement of tissue grinding/liquid nitrogen/hazardous chemicals and above all, equally effective for both fresh and old samples. Using this method, costs per prep comes down ~ 10-50 times as compared to most commercial kits. This method can be used for on-field experiments like molecular diagnostics, varietal identification etc.

Dataset Information

A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes.

Publications

A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes.

Similar Datasets

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