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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsAbbreviations
- 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
References
Tiollais P, Pourcel C, Dejean A (1985) The hepatitis B virus. Nature 317(6037):489–495
World Health Organization (1997) The World Health Report 1997
World Health Organization (2007) Hepatitis B. World Health Organization Fact Sheet 204 (Revised October 2000)
Seeger C, Mason WS (2000) Hepatitis B virus biology. Microbiol Mol Biol Rev 64(1):51–68
Ganem D, Schneider RJ (2002) Hapdnaviridae: the viruses and their replication. In: Knipe DM, Howley PM (eds) Fields virology, 4th edn. Wolters Kluwer, The Netherlands, pp 2923–2968
Tuttleman JS, Pourcel C, Summers J (1986) Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 47(3):451–460
Newbold JE, Xin H, Tencza M, Sherman G, Dean J, Bowden S et al (1995) The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes. J Virol 69(6):3350–3357
Singh M, Dicaire A, Wakil AE, Luscombe C, Sacks SL (2004) Quantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in the liver of HBV-infected patients by LightCycler real-time PCR. J Virol Methods 118(2):159–167
Werle-Lapostolle B, Bowden S, Locarnini S, Wursthorn K, Petersen J, Lau G et al (2004) Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology 126(7):1750–1758
Wursthorn K, Lutgehetmann M, Dandri M, Volz T, Buggisch P, Zollner B et al (2006) Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B. Hepatology 44(3):675–684
Laras A, Koskinas J, Dimou E, Kostamena A, Hadziyannis SJ (2006) Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients. Hepatology 44(3):694–702
Volz T, Lutgehetmann M, Wachtler P, Jacob A, Quaas A, Murray JM et al (2007) Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients. Gastroenterology 133(3):843–852
Bourne EJ, Dienstag JL, Lopez VA, Sander TJ, Longlet JM, Hall JG et al (2007) Quantitative analysis of HBV cccDNA from clinical specimens: correlation with clinical and virological response during antiviral therapy. J Viral Hepat 14(1):55–63
Sung JJ, Wong ML, Bowden S, Liew CT, Hui AY, Wong VW et al (2005) Intrahepatic hepatitis B virus covalently closed circular DNA can be a predictor of sustained response to therapy. Gastroenterology 128(7):1890–1897
Wong DK, Yuen MF, Yuan H, Sum SS, Hui CK, Hall J et al (2004) Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients. Hepatology 40(3):727–737
Mazet-Wagner AA, Baclet MC, Loustaud-Ratti V, Denis F, Alain S (2006) Real-time PCR quantitation of hepatitis B virus total DNA and covalently closed circular DNA in peripheral blood mononuclear cells from hepatitis B virus-infected patients. J Virol Methods 138(1–2):70–79
Chen CJ, Yang HI, Su J, Jen CL, You SL, Lu SN et al (2006) Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA 295(1):65–73
Yuen MF, Wong DK, Sum SS, Yuan HJ, Yuen JC, Chan AO et al (2005) Effect of lamivudine therapy on the serum covalently closed-circular (ccc) DNA of chronic hepatitis B infection. Am J Gastroenterol 100(5):1099–1103
Boom R, Sol CJ, Heijtink R, Wertheim-van Dillen PM, van der NJ (1991) Rapid purification of hepatitis B virus DNA from serum. J Clin Microbiol 29(9):1804–1811
Boom R, Sol C, Beld M, Weel J, Goudsmit J, Wertheim-van DP (1999) Improved silica-guanidiniumthiocyanate DNA isolation procedure based on selective binding of bovine alpha-casein to silica particles. J Clin Microbiol 37(3):615–619
Boom R, Sol C, Weel J, Gerrits Y, de BM, Wertheim-van DP (1999) A highly sensitive assay for detection and quantitation of human cytomegalovirus DNA in serum and plasma by PCR and electrochemiluminescence. J Clin Microbiol 37(5):1489–1497
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media New York
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-61779-937-2_7
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-936-5
Online ISBN: 978-1-61779-937-2
eBook Packages: Springer Protocols