title: Nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA.
abstract: BACKGROUND
  Successful treatment of hepatitis B can be achieved only if the template for hepatitis B virus (HBV) DNA replication, the covalently closed circular HBV DNA (cccDNA) can be completely cleared. To date, detecting cccDNA remains clinically challenging. The purpose of this study was to develop a nested real-time quantitative polymerase chain reaction (PCR) assay for detecting HBV cccDNA in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (MMNCs).
  
  
  METHODS
  Based on the structural differences between HBV cccDNA and HBV relaxed circular DNA (rcDNA), two pairs of primers were synthesized as well as a downstream TaqMan probe. Blood and bone marrow samples were collected from hepatitis B patients and healthy controls. To remove rcDNA, samples were incubated with mung bean nuclease and the resultant purified HBV cccDNA was then amplified by nested real-time fluorescence quantitative PCR. The cccDNA levels were calculated using a positive standard.
  
  
  RESULTS
  The nested real-time fluorescence quantitative PCR method for HBV cccDNA was successful, with a linear range of 3.0 × 10(2) copies/ml to 3.9 × 10(8) copies/ml. Of the 25 PBMC samples and 7 MMNC samples obtained from chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were positive for HBV cccDNA, while all of the 21 PBMC samples from healthy controls were negative.
  
  
  CONCLUSIONS
  The nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.
authors: Department of Infectious Diseases, Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150086, China.
authors: Chun-Hai
authors: CH
authors: Xu
authors: Zhao-Shen
authors: ZS
authors: Li
authors: Jun-Ying
authors: JY
authors: Dai
authors: Hai-Yang
authors: HY
authors: Zhu
authors: Jian-Wu
authors: JW
authors: Yu
authors: Shu-Lan
authors: SL
authors: Lü
mesh_terms: D002478:Cells, Cultured; D004270:DNA, Circular / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral