Hepatitis B virus DNA quantification with the three-in-one (3io) method allows accurate single-step differentiation of total HBV DNA and cccDNA in biopsy-size liver samples☆,☆☆,☆☆☆

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Abstract

Background

Hepatitis B virus (HBV) replicates via reverse transcription converting its partially double stranded genome into the covalently closed circular DNA (cccDNA). The long-lasting cccDNA serves as a replication intermediate in the nuclei of hepatocytes. It is an excellent, though evasive, parameter for monitoring the course of liver disease and treatment efficiency.

Objective

To develop and test a new approach for HBV DNA quantification in serum and small-size liver samples.

Study design

The p3io plasmid contains an HBV fragment and human β-actin gene (hACTB) as a standard. Respective TaqMan probes were labeled with different fluorescent dyes. A triplex real-time PCR for simultaneous quantification of total HBV DNA, cccDNA and hACTB could be established.

Results

Three-in-one method allows simultaneous analysis of 3 targets with a lower limit of quantification of 48 copies per 20 μl PCR reaction and a wide range of linearity (R2 > 0.99, p < 0.0001) for all measured sequences. The method showed a pan-genotypic specificity among genotypes A–F with serum DNA samples from HBV infected patients. Total HBV DNA and cccDNA could be quantified in 32 and 22 of 33 FFPE preserved liver specimens, respectively. Total HBV DNA concentrations quantified by the 3io method remained comparable with Cobas TaqMan HBV Test v2.0.

Conclusions

The three-in-one protocol allows the single step quantification of viral DNA in samples from different sources. Therefore lower sample input, faster data acquisition, a lowered error and significantly lower costs are the advantages of the method.

Section snippets

Background

Hepatitis B virus (HBV), the eponymous member of the Hepandnaviridae family causes 240 million chronic infections worldwide [1], [2]. Persistent viral infection results in increasing risk of liver fibrosis, cirrhosis and hepatocellular carcinoma [3]. Efficient vaccination has been available since the 1980s and reduced the incidence in countries that have adopted a HBV vaccination program [4], [5], [6]. Despite these efforts HBV infection and its squeal remain a global challenge. It is estimated

Objectives

Measuring serum HBV markers does not fully reflect the status of the infection in hepatocytes [33]. Liver tissue from biopsies is usually evaluated for the grading of inflammation and the staging of fibrosis but not viral genomic DNA and replication intermediates [25], [33]. Intrahepatic HBV DNA quantification can be an ideal tool for the study of viral infection status and efficiency of antiviral treatment [21], [25], [26], [27], [39]. The small amount of tissue limits the routine application

Plasmid construction and preparation of plasmid stock solutions

The plasmid was cloned serving as quantification standard. An HBV fragment and hACTB exon 4 as genome copy normalization gene were inserted into the pUC19 (Invitrogen) cloning vector. The 1945 bp HBV fragment corresponding nucleotides 1201–3145 in reference genome (GenBank accession number: V01460) was derived from a genotype D HBV plasmid and cloned into an EcoRI restriction site. A 395 bp hACTB fragment from exon 4 was amplified using High Fidelity PCR Master (Roche) with primers carrying Hin

Specificity and sensitivity of the 3io method

PCR products of distinct samples were confirmed on a gel (Fig. 2). Serial dilutions of the p3io plasmid ranging from 4.8 × 10 to 4.8 × 106 cps/rxn gave good quantification signals in all channels. To determine further specificity, commercially available mouse and human genomic DNA (Promega) and DNA from RAW264.7 and Msh cell lines were tested. The two HBV channels (FAM, Cy5) were negative and hACTB channel (Cy3) was positive with human DNA and negative for murine Msh and RAW264.7 cell lines.

Discussion

Different PCR methods were previously designed to quantify total HBV DNA and cccDNA concentrations from liver tissue samples with the need for a separate qPCR for the normalization of the housekeeping gene [25], [29], [40], [41].

Here we established a novel approach for the simultaneous quantification of total HBV DNA, cccDNA and hACTB for DNA normalization from small amounts of liver tissue using a triplex real-time PCR. We could show that the ability to simultaneously quantify 3 targets in one

Funding

German Centre for Infection Research (DZIF found number 19360823 and 19360917). BTS was supported by a scholarship from project 322 of Vietnam Ministry of Education and Training, Vietnam.

Competing interests

None declared.

Ethical approval

Not required.

Authors’ contribution

AT, SBT – method conception and design, data acquisition, analysis and interpretation, drafting the article; BJZ – data acquisition, sequences analysis, critical revision of the draft; MR-T – data acquisition, critical revision of the draft; CThB – provided plasmid pHBV13, MPM, CThB, KW – interpretation, drafting the article, approval of the submitted version of the article.

Acknowledgments

We would like to thank Prof. Dr. J. Engelbert Geßner and Georgios A. Sogkas from Clinic for Immunology and Rheumatology, Hannover Medical School for providing us DNA from RAW264.7 and Msh mouse macrophage cell lines, Dr. Thomas von Hahn and Katrin Rohrmann from Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School for providing Hep38AD cells, and Patrick Lehmann, Martina Darnedde, and Birgit Bremer from Hepsero Routin Diagnostic Laboratory. Our thanks we address

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    • ☆

    This work was supported by founds 19360823 and 19360917 from German Centre for Infection Research (DZIF).

    ☆☆

    AT is a student of the PhD Program Infection Biology at the Center for Infection Biology (ZIB) and the Hannover Biomedical Research School (HBRS).

    ☆☆☆

    BTS was supported by a scholarship from project 322 of Vietnam Ministry of Education and Training, Vietnam.

    1

    These authors contributed equally to this work.

    2

    These authors contributed equally to this work.

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