title: Quantitation of HBV covalently closed circular DNA in micro formalin fixed paraffin-embedded liver tissue using rolling circle amplification in combination with real-time PCR.
abstract: BACKGROUND
  The study aimed to develop an effective method to quantitate HBV covalently closed circular DNA (cccDNA) using small section of formalin fixed paraffin-embedded (FFPE) liver biopsy.
  
  
  METHODS
  Plasmid-safe ATP-dependent DNase (PSAD)-treated samples were subjected to rolling circle amplification (RCA) prior to real-time PCR mediated by cccDNA-selective primers. Human beta-actin gene was used as a reference control.
  
  
  RESULTS
  Compared to the classical method, i.e., PSAD digestion+real-time PCR, introduction of RCA increased the lower limit of detection for about 2 logs with good inter- and intra-assay reproducibility. HBV cccDNA was detected in 91.5% (119/130) of the FFPE samples. The cccDNA levels (copy/cell) between FFPE liver tissues and fresh frozen counterpart tissues were comparable. The median of cccDNA level in HBeAg-positive patients was higher than that in HBeAg-negative ones (52.60 vs. 31.25copies/cell, P<0.01). Intrahepatic cccDNA level was positively correlated with intrahepatic HBV total DNA level, but not obviously correlated with serum HBV DNA or alanine aminotransferase levels.
  
  
  CONCLUSIONS
  The method could sensitively and specifically quantitate intrahepatic HBV cccDNA in micro FFPE liver biopsy tissue for evaluation of HBV replication status in the liver.
authors: Institution of Infectious Diseases, Beijing 302 Hospital, Beijing, China.
authors: Yanwei
authors: Y
authors: Zhong
authors: Jiaqi
authors: J
authors: Han
authors: Zhengsheng
authors: Z
authors: Zou
authors: Shuhong
authors: S
authors: Liu
authors: Bo
authors: B
authors: Tang
authors: Xiaoqiang
authors: X
authors: Ren
authors: Xiaodong
authors: X
authors: Li
authors: Yulai
authors: Y
authors: Zhao
authors: Yan
authors: Y
authors: Liu
authors: Dongqing
authors: D
authors: Zhou
authors: Weijie
authors: W
authors: Li
authors: Jingmin
authors: J
authors: Zhao
authors: Dongping
authors: D
authors: Xu
mesh_terms: D004270:DNA, Circular / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D005260:Female; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000473:pathology* / Q000821:virology; D008297:Male; D016612:Paraffin Embedding / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral