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.
  
  
  METHODS
  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 (R(2)>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.
authors.affiliation: Hannover Medical School (MHH), Department of Gastroenterology, Hepatology and Endocrinology, Carl-Neuberg-Straße 1, 30623 Hannover, Germany.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Taranta
authors.affiliation: Robert Koch Institute, Department of Infectious Diseases, Seestraße 10, 13353 Berlin, Germany; Institute of Tropical Medicine, University of Tuebingen, Wilhelmstraße 27D, 72074 Tübingen, Germany.
authors.forename: Bui
authors.initials: B
authors.lastname: Tien Sy
authors.affiliation: Hannover Medical School (MHH), Department of Gastroenterology, Hepatology and Endocrinology, Carl-Neuberg-Straße 1, 30623 Hannover, Germany.
authors.forename: Behrend Johan
authors.initials: BJ
authors.lastname: Zacher
authors.affiliation: Hannover Medical School (MHH), Department of Gastroenterology, Hepatology and Endocrinology, Carl-Neuberg-Straße 1, 30623 Hannover, Germany; Department of Infectious Diseases and Hepatology, Medical University of Bialystok, Żurawia 14, 15540 Białystok, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Rogalska-Taranta
authors.affiliation: Hannover Medical School (MHH), Department of Gastroenterology, Hepatology and Endocrinology, Carl-Neuberg-Straße 1, 30623 Hannover, Germany.
authors.forename: Michael Peter
authors.initials: MP
authors.lastname: Manns
authors.affiliation: Robert Koch Institute, Department of Infectious Diseases, Seestraße 10, 13353 Berlin, Germany.
authors.forename: Claus Thomas
authors.initials: CT
authors.lastname: Bock
authors.affiliation: Hannover Medical School (MHH), Department of Gastroenterology, Hepatology and Endocrinology, Carl-Neuberg-Straße 1, 30623 Hannover, Germany; ifi - Institut für Interdisziplinäre Medizin, Lohmühlenstraße. 5, 20099 Hamburg, Germany. Electronic address: wursthorn@gmail.com.
authors.forename: Karsten
authors.initials: K
authors.lastname: Wursthorn
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000097:blood; D045744:Cell Line, Tumor; D004270:DNA, Circular / Q000097:blood*; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D005838:Genotype; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000145:classification / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000097:blood* / Q000175:diagnosis / Q000235:genetics; D006801:Humans; D008099:Liver / Q000821:virology*; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods; D014021:Tissue Preservation
chemical_list: D015415:Biomarkers; D004270:DNA, Circular; D004279:DNA, Viral
keywords: FFPE; HBV DNA; HBsAg; Quantification; Real-time PCR; cccDNA