title: [Establishment and application of nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA].
abstract: OBJECTIVE
  To establish a nested real-time quantitative polymerase chain reaction (PCR) assay for detection of hepatitis B virus covalently closed circular DNA in PBMC( peripheral blood monocyte) and MMNC (marrow monocyte).
  
  
  METHODS
  Based on the structural differences between HBVcccDNA and HBV rcDNA, two pairs of specific primers spanned the gap of the positive and negative chains and a specific TaqMan probe situated downstream were designed. To remove rcDNA, cccDNA was processed by Mung Bean Nuclease,and then amplified by nested real-time quantitative PCR using a pair of outer primers and a pair of inner primers. According to the standard preparation, cccDNA levels of specimen were calculated.
  
  
  RESULTS
  We have established a nested real-time fluorescent quantitative PCR method for HBV cccDNA successfully, and the linear range is from 5.0 x 10(2) to 3. 9 x 10(7) copies per milliliter. Of the 25 PBMC samples and 7 MMNC samples of the chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were HBV cccDNA positive, while all of the 21 healthy donator blood PBMC samples were negative.
  
  
  CONCLUSIONS
  The nested real-time fluorescent quantitative PCR method may be applied to detect HBVcccDNA level in PBMC and MMNC. HBVcccDNA can be detected in PBMC and MMNC.
authors.affiliation: The Second Affiliated Hospital of Harbin Medical University, 150086, China.
authors.forename: Chun-hai
authors.initials: CH
authors.lastname: Xu
authors.forename: Zhao-shen
authors.initials: ZS
authors.lastname: Li
authors.forename: Jun-ying
authors.initials: JY
authors.lastname: Dai
authors.forename: Hai-yang
authors.initials: HY
authors.lastname: Zhu
authors.forename: Jian-wu
authors.initials: JW
authors.lastname: Yu
authors.forename: Shu-lan
authors.initials: SL
authors.lastname: Lv
mesh_terms: D004270:DNA, Circular / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D006509:Hepatitis B / Q000175:diagnosis / Q000821:virology*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral