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

Andrzej Taranta; Bui Tien Sy; Behrend Johan Zacher; Magdalena Rogalska-Taranta; Michael Peter Manns; Claus Thomas Bock; Karsten Wursthorn

doi:10.1016/j.jcv.2014.04.015

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

Andrzej Taranta, Bui Tien Sy, Behrend Johan Zacher, Magdalena Rogalska-Taranta, Michael Peter Manns, Claus Thomas Bock, Karsten Wursthorn^*

^*Corresponding author for this work

6 Citations (Scopus)

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 20μl 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.

Original language	English
Journal	Journal of Clinical Virology
Volume	60
Issue number	4
Pages (from-to)	354-360
Number of pages	7
ISSN	1386-6532
DOIs	https://doi.org/10.1016/j.jcv.2014.04.015
Publication status	Published - 1 Jan 2014
Externally published	Yes

Keywords

CccDNA
FFPE
HBsAg
HBV DNA
Quantification
Real-time PCR

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.jcv.2014.04.015

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Taranta, A., Tien Sy, B., Zacher, B. J., Rogalska-Taranta, M., Manns, M. P., Bock, C. T., & Wursthorn, K. (2014). 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. Journal of Clinical Virology, 60(4), 354-360. https://doi.org/10.1016/j.jcv.2014.04.015

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. / Taranta, Andrzej; Tien Sy, Bui; Zacher, Behrend Johan et al.

In: Journal of Clinical Virology, Vol. 60, No. 4, 01.01.2014, p. 354-360.

Research output: Contribution to journal › Journal article › Research › peer-review

Taranta, A, Tien Sy, B, Zacher, BJ, Rogalska-Taranta, M, Manns, MP, Bock, CT & Wursthorn, K 2014, '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', Journal of Clinical Virology, vol. 60, no. 4, pp. 354-360. https://doi.org/10.1016/j.jcv.2014.04.015

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title = "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",

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 20μl 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.",

keywords = "CccDNA, FFPE, HBsAg, HBV DNA, Quantification, Real-time PCR",

author = "Andrzej Taranta and {Tien Sy}, Bui and Zacher, {Behrend Johan} and Magdalena Rogalska-Taranta and Manns, {Michael Peter} and Bock, {Claus Thomas} and Karsten Wursthorn",

year = "2014",

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doi = "10.1016/j.jcv.2014.04.015",

language = "English",

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T1 - 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

AU - Taranta, Andrzej

AU - Tien Sy, Bui

AU - Zacher, Behrend Johan

AU - Rogalska-Taranta, Magdalena

AU - Manns, Michael Peter

AU - Bock, Claus Thomas

AU - Wursthorn, Karsten

PY - 2014/1/1

Y1 - 2014/1/1

N2 - 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 20μl 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.

AB - 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 20μl 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.

KW - CccDNA

KW - FFPE

KW - HBsAg

KW - HBV DNA

KW - Quantification

KW - Real-time PCR

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SN - 1386-6532

VL - 60

SP - 354

EP - 360

JO - Journal of Clinical Virology

JF - Journal of Clinical Virology

IS - 4

ER -

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

Abstract

Keywords

UN SDGs

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