Quantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in the liver of HBV-infected patients by LightCycler™ real-time PCR

被引:46
作者
Singh, M
Dicaire, A
Wakil, AE
Luscombe, C
Sacks, SL
机构
[1] Viridae Clin Sci Inc, Vancouver, BC V6Z 1Y8, Canada
[2] Calif Pacific Med Ctr, San Francisco, CA 94120 USA
[3] Univ British Columbia, Fac Med, Dept Pharmacol & Therapeut, Vancouver, BC V6T 1Z3, Canada
关键词
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.
引用
收藏
页码:159 / 167
页数:9
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