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Validation of a Sensitive and Specific Real-Time PCR for Detection and Quantitation of Hepatitis B Virus Covalently Closed Circular DNA in Plasma of Chronic Hepatitis B Patients

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Diagnosis of Sexually Transmitted Diseases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 903))

Abstract

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) serves as a template for viral replication and plays a role in persistence of HBV infection. The origin and significance of cccDNA in plasma, however, are not well understood. A sensitive, specific, and reproducible real-time PCR for detection and quantitation of cccDNA in plasma of chronic hepatitis B patients was developed and validated. Four HBV DNA reference panels and 96 plasma samples of chronic hepatitis B patients are analyzed. Results are compared with total HBV DNA levels. This cccDNA assay had a lower limit of detection at 15 copies/PCR, a lower limit of quantitation at 91 copies/PCR, and a correlation coefficient (R) of 0.98 (p < 0.0001). HBV cccDNA can be detected in two of four international panels. Significant correlation is found between cccDNA and total HBV DNA levels in both panels (R = 0.96 and R = 0.43) and in samples of the chronic hepatitis B patients (R = 0.88, p < 0.0001). In 57 % of these samples cccDNA can be detected. Mean level of cccDNA is 0.16 % of total HBV load. Plasma HBV cccDNA levels are higher in HBeAg-positive samples than in HBeAg-negative samples (p < 0.0001). Total HBV DNA levels and HBV genotype do not influence cccDNA detection.

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Abbreviations

HBV:

Hepatitis B virus

cccDNA:

Covalently closed circular DNA

ALT:

Alanine transaminase

HAI:

Histology activity index

PCR:

Polymerase chain reaction

BSA:

Bovine serum albumin

q-PCR:

Quantitative real-time PCR

SC:

Silica particles

SiO2 :

Silicon dioxide

GuSCN:

Guanidinium thiocyanate

HCl:

Hydrochloric acid

EDTA:

Ethylenediaminetetraacetic acid

Tris buffer:

Trisamine (hydroxymethylaminoethane) buffer

TE buffer:

Tris/HCl/EDTA buffer

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Authors and Affiliations

  1. Department of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

    R. B. Takkenberg

  2. Biomedical Research, Royal Tropical Institute, Amsterdam, The Netherlands

    S. Menting & M. G. H. M. Beld

Authors
  1. R. B. Takkenberg
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  2. S. Menting
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  3. M. G. H. M. Beld
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Corresponding author

Correspondence to M. G. H. M. Beld .

Editor information

Editors and Affiliations

  1. University Hospital, Institute of Medical Microbiology, Heinrich-Heine-University, Universitätssraße 1, Düsseldorf, 40225, Germany

    Colin R. MacKenzie

  2. University Hospital, Institute of Medical Microbiology, Heinrich-Heine-University, Universitätssraße 1, Düsseldorf, 40225, Germany

    Birgit Henrich

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© 2012 Springer Science+Business Media New York

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Takkenberg, R.B., Menting, S., Beld, M.G.H.M. (2012). Validation of a Sensitive and Specific Real-Time PCR for Detection and Quantitation of Hepatitis B Virus Covalently Closed Circular DNA in Plasma of Chronic Hepatitis B Patients. In: MacKenzie, C., Henrich, B. (eds) Diagnosis of Sexually Transmitted Diseases. Methods in Molecular Biology, vol 903. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-937-2_7

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  • DOI: https://doi.org/10.1007/978-1-61779-937-2_7

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-936-5

  • Online ISBN: 978-1-61779-937-2

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