Primer designing for PreS region of hepatitis B virus from the most conserved patches of hepatitis B virus genome

Abbas, Naaz; Ajmal, M. Asir; Afroze, Talat

doi:10.5897/jbsa2011.021

Cited by 2 publications

(1 citation statement)

References 16 publications

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“…Briefly, the pre-S1 through S regions were amplified using conserved universal primers (5'-TCACCATATTCTTGGGAACAAGA-3'and 5'-CGAAC CACTGAACAAATGGC-3'), and HBV genotypes were detected based on sequencing by Bionics (Seoul, Korea). An HBV-specific PreS primer [11] (5'-CCCTAGAAAATTGAGAGAAGTCCA-3') was used for sequencing. The sequences obtained were compared with published sequences covering the same genomic region of 8 HBV genotypes, as available in the National Center for Biotechnology Information GenBank.…”

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

Yang

Koenig

et al. 2020

Viruses

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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.

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mentioning

confidence: 99%

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

Yang

Koenig

et al. 2020

Viruses

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Traveler’s Infections: Overview of Hepatitis B Virus Infection

Oti¹

2021

Tourism

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Hepatitis B virus (HBV) is a double-stranded circular DNA virus that infects the hepatocytes. HBV infection is considered as an important public health concern globally especially with one-third of the world's population been infected. Local and international migrants are one of those population at high risk of the infection. Many factors interplay in the acquisition of HBV such as purpose of travel, destination endemicity rate of the virus, time of stay of the traveler, inadequate prevention and control measures, among others, understanding the genotypes of HBV is critical in correlating the evolution of the virus and migration of humans and also treatment responses of infected population. The symptom of the virus ranges from fever to jaundice and to a liver cirrhosis and hepatocellular carcinoma (HCC). Transmission of HBV is commonly via horizontal route in developing regions and in the developed regions; transmission occur more often among adults that use injectable drugs and high-risk sexual behaviors. Therefore, the Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) have recommended HBV screening and vaccination to all travelers without an HBV immunization history before traveling to endemic regions. This chapter gives an overview on HBV as a potential traveler's infection.

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Primer designing for PreS region of hepatitis B virus from the most conserved patches of hepatitis B virus genome

Cited by 2 publications

References 16 publications

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

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

Traveler’s Infections: Overview of Hepatitis B Virus Infection

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