title: [A quantitative real time polymerase chain reaction for detection of HBV covalently closed circular DNA in livers of the HBV infected patients].
abstract: OBJECTIVE
  To establish and optimize a sensitive and specific quantitative real-time polymerase chain reaction (PCR) method for detection of hepatitis B virus covalently closed circular DNA (HBV cccDNA) in liver tissue.
  
  
  METHODS
  Specific primers and probes were designed to detect HBV DNA (tDNA) and cccDNA. A series of plasmids (3.44 × 10(0) - 3.44 × 10(9) copies/µl) containing a full double-stranded copies of HBV genome (genotype C) were used to establish the standard curve of real-time PCR. Liver samples of 33 patients with HBV related hepatocellular carcinoma (HCC), 13 Chronic hepatitis B patients (CHB) and 10 non-HBV patients were collected to verify the sensitivity and specificity of the assay. A fraction of extracted DNA was digested with a Plasmid-Safe ATP-dependent Dnase (PSAD) for HBV cccDNA detection and the remaining was used for tDNA and β-globin detection. The amount (copies/cell) of HBV cccDNA and tDNA were measured by a real-time PCR, using β-globin housekeeping gene as a quantitation standard.
  
  
  RESULTS
  The standard curves of real-time PCR with a linear range of 3.44 × 10(0) to 3.44 × 10(9) copies/µl were established for detecting HBV cccDNA and tDNA, and both of the lowest detection limits of HBV cccDNA and tDNA were 3.44 × 10(0) copies/µl. The lowest quantitation levels of HBV cccDNA in liver tissues tested in 33 HBV related HCC patients and 13 CHB patients were 0.003 copies/cell and 0.031 copies/cell, respectively. HBV cccDNA and tDNA in liver tissue of 10 non-HBV patient appeared to be negative. The true positive rate was increasing through the digestion of HBV DNA by PSAD, and the analytic specificity of cccDNA detection improved by 7.24 × 10(2) times. Liver tissues of 2 patients were retested 5 times in the PCR for detecting cccDNA and the coefficient of variations on cycle threshold (Ct) were between 0.224% - 0.609%.
  
  
  CONCLUSIONS
  A highly sensitive and specific quantitative real time PCR method for the detection of HBV cccDNA in liver tissue was established and could be used for clinical and epidemiological studies.
authors.affiliation: Department of Microbiology, Peking University Health Science Center, Beijing 100191, China.
authors.forename: Mei-Rong
authors.initials: MR
authors.lastname: Wang
authors.forename: Ning
authors.initials: N
authors.lastname: Qiu
authors.forename: Shi-Chun
authors.initials: SC
authors.lastname: Lu
authors.forename: Dian-Rong
authors.initials: DR
authors.lastname: Xiu
authors.forename: Jian-Guo
authors.initials: JG
authors.lastname: Yu
authors.forename: Tong
authors.initials: T
authors.lastname: Li
authors.forename: Xue-En
authors.initials: XE
authors.lastname: Liu
authors.forename: Hui
authors.initials: H
authors.lastname: Zhuang
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D017931:DNA Primers; D004270:DNA, Circular / Q000302:isolation & purification*; D004279:DNA, Viral / Q000302:isolation & purification*; D005260:Female; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000821:virology; D008297:Male; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D055815:Young Adult
chemical_list: D017931:DNA Primers; D004270:DNA, Circular; D004279:DNA, Viral