hepatitis B virus;
cccDNA;
LightCycler;
real-time PCR;
D O I:
10.1016/j.jviromet.2004.02.006
中图分类号:
Q5 [生物化学];
学科分类号:
071010 ;
081704 ;
摘要:
Antivirals for hepatitis B virus (HBV) reduce viral load and improve liver histology, however, their effect on covalently closed circular DNA (cccDNA), the HBV transcriptional template, has not been extensively examined. This study evaluated a newly designed LightCycler(TM)-based quantitative cccDNA PCR assay. A linear range of 2.5 x 10(1) to 1 x 10(9) copies/assay using primers specific for HBV cccDNA and 2.5 x 10(1) to 2 x 10(9) copies/assay using primers specific for total HBV DNA (tDNA) was established. beta-Globin was used to estimate the number of cells in each PCR reaction. Enzymatic digestion with an ATP-dependent DNase improved the analytic specificity to a greater than 1: 10,000 ratio of cccDNA:RC DNA (relaxed circular DNA). One-tenth of the extracted DNA from 1 mg of liver biopsy, was analyzed from six patients, three HBV-infected and three uninfected individuals, under blinded conditions; three were found positive and three negative for cccDNA and tDNA. Approximately 6 x 10(3) copies of cccDNA/mg of tissue were detected in a pre-transplant biopsy from an HBV-infected patient treated with lamivudine. Sequential post-transplant liver biopsies were negative for both HBV cccDNA and tDNA. An HBV-infected patient with cirrhosis who was antiviral therapy naive had 3.7 x 10(4) copies of cccDNA/mg of liver tissue. Another treatment-naive patient with a history of high HBV viral load had 1 x 10(5) copies of cccDNA/mg of tissue (4 x 10(6) copies of tDNA/mg of tissue). Further studies are warranted but the high level of sensitivity, specificity, rapidity and accuracy provided by this novel assay with the LightCycler(TM) system indicate that it could be useful for monitoring antiviral therapy. (C) 2004 Elsevier B.V. All rights reserved.
机构:
Inst Mol Technol Drug Discovery & Synth, Inst Mol Biol, Hong Kong, Hong Kong, Peoples R ChinaInst Mol Technol Drug Discovery & Synth, Inst Mol Biol, Hong Kong, Hong Kong, Peoples R China
He, ML
;
Wu, J
论文数: 0引用数: 0
h-index: 0
机构:Inst Mol Technol Drug Discovery & Synth, Inst Mol Biol, Hong Kong, Hong Kong, Peoples R China
Wu, J
;
Chen, Y
论文数: 0引用数: 0
h-index: 0
机构:Inst Mol Technol Drug Discovery & Synth, Inst Mol Biol, Hong Kong, Hong Kong, Peoples R China
Chen, Y
;
Lin, MC
论文数: 0引用数: 0
h-index: 0
机构:Inst Mol Technol Drug Discovery & Synth, Inst Mol Biol, Hong Kong, Hong Kong, Peoples R China
Lin, MC
;
Lau, GKK
论文数: 0引用数: 0
h-index: 0
机构:Inst Mol Technol Drug Discovery & Synth, Inst Mol Biol, Hong Kong, Hong Kong, Peoples R China
Lau, GKK
;
Kung, HF
论文数: 0引用数: 0
h-index: 0
机构:Inst Mol Technol Drug Discovery & Synth, Inst Mol Biol, Hong Kong, Hong Kong, Peoples R China
机构:Univ Hong Kong, Queen Mary Hosp, Dept Med, Hong Kong, Hong Kong, Peoples R China
Ho, SKN
;
Yam, WC
论文数: 0引用数: 0
h-index: 0
机构:Univ Hong Kong, Queen Mary Hosp, Dept Med, Hong Kong, Hong Kong, Peoples R China
Yam, WC
;
Leung, ETK
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机构:Univ Hong Kong, Queen Mary Hosp, Dept Med, Hong Kong, Hong Kong, Peoples R China
Leung, ETK
;
Wong, LP
论文数: 0引用数: 0
h-index: 0
机构:Univ Hong Kong, Queen Mary Hosp, Dept Med, Hong Kong, Hong Kong, Peoples R China
Wong, LP
;
Leung, JKH
论文数: 0引用数: 0
h-index: 0
机构:Univ Hong Kong, Queen Mary Hosp, Dept Med, Hong Kong, Hong Kong, Peoples R China
Leung, JKH
;
Lai, KN
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机构:Univ Hong Kong, Queen Mary Hosp, Dept Med, Hong Kong, Hong Kong, Peoples R China
Lai, KN
;
Chan, TM
论文数: 0引用数: 0
h-index: 0
机构:
Univ Hong Kong, Queen Mary Hosp, Dept Med, Hong Kong, Hong Kong, Peoples R ChinaUniv Hong Kong, Queen Mary Hosp, Dept Med, Hong Kong, Hong Kong, Peoples R China
机构:
Inst Mol Technol Drug Discovery & Synth, Inst Mol Biol, Hong Kong, Hong Kong, Peoples R ChinaInst Mol Technol Drug Discovery & Synth, Inst Mol Biol, Hong Kong, Hong Kong, Peoples R China
He, ML
;
Wu, J
论文数: 0引用数: 0
h-index: 0
机构:Inst Mol Technol Drug Discovery & Synth, Inst Mol Biol, Hong Kong, Hong Kong, Peoples R China
Wu, J
;
Chen, Y
论文数: 0引用数: 0
h-index: 0
机构:Inst Mol Technol Drug Discovery & Synth, Inst Mol Biol, Hong Kong, Hong Kong, Peoples R China
Chen, Y
;
Lin, MC
论文数: 0引用数: 0
h-index: 0
机构:Inst Mol Technol Drug Discovery & Synth, Inst Mol Biol, Hong Kong, Hong Kong, Peoples R China
Lin, MC
;
Lau, GKK
论文数: 0引用数: 0
h-index: 0
机构:Inst Mol Technol Drug Discovery & Synth, Inst Mol Biol, Hong Kong, Hong Kong, Peoples R China
Lau, GKK
;
Kung, HF
论文数: 0引用数: 0
h-index: 0
机构:Inst Mol Technol Drug Discovery & Synth, Inst Mol Biol, Hong Kong, Hong Kong, Peoples R China
机构:Univ Hong Kong, Queen Mary Hosp, Dept Med, Hong Kong, Hong Kong, Peoples R China
Ho, SKN
;
Yam, WC
论文数: 0引用数: 0
h-index: 0
机构:Univ Hong Kong, Queen Mary Hosp, Dept Med, Hong Kong, Hong Kong, Peoples R China
Yam, WC
;
Leung, ETK
论文数: 0引用数: 0
h-index: 0
机构:Univ Hong Kong, Queen Mary Hosp, Dept Med, Hong Kong, Hong Kong, Peoples R China
Leung, ETK
;
Wong, LP
论文数: 0引用数: 0
h-index: 0
机构:Univ Hong Kong, Queen Mary Hosp, Dept Med, Hong Kong, Hong Kong, Peoples R China
Wong, LP
;
Leung, JKH
论文数: 0引用数: 0
h-index: 0
机构:Univ Hong Kong, Queen Mary Hosp, Dept Med, Hong Kong, Hong Kong, Peoples R China
Leung, JKH
;
Lai, KN
论文数: 0引用数: 0
h-index: 0
机构:Univ Hong Kong, Queen Mary Hosp, Dept Med, Hong Kong, Hong Kong, Peoples R China
Lai, KN
;
Chan, TM
论文数: 0引用数: 0
h-index: 0
机构:
Univ Hong Kong, Queen Mary Hosp, Dept Med, Hong Kong, Hong Kong, Peoples R ChinaUniv Hong Kong, Queen Mary Hosp, Dept Med, Hong Kong, Hong Kong, Peoples R China