期刊
JOURNAL OF VIROLOGICAL METHODS
卷 169, 期 1, 页码 181-187出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jviromet.2010.07.031
关键词
Hepatitis B virus; cccDNA; Quantitation; Real-time PCR; Restriction endonuclease; Plasmid-safe ATP-dependent DNase
资金
- Key Science and Technology Project of Tianjin [05YFSZSF02500]
- Applied Basic Research Foundation of Tianjin [06YFJMJC13100]
- National Basic Research Program of China [2007CB512800]
The persistence of covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) in hepatocytes plays a key role in viral persistence and resistance to therapy. Therefore, quantitative cccDNA measurement is of clinical importance for evaluating the efficacy of antiviral drugs, selecting an appropriate treatment strategy, and predicting the prognosis. Current established methods for measurement of cccDNA need further improvement. A modified method was developed using digestion with restriction endonucleases that do not recognize sites in the HBV DNA and plasmid-safe ATP-dependent DNase (PSAD), and using a cccDNA-specific primer set in a real-time PCR reaction. The cccDNA-specific primer has a similar amplification efficiency as a commercial kit. Treatment of samples with restriction endonuclease followed by PSAD digestion increased significantly the specificity of a cccDNA-selective primer set compared with other treatments (P < 0.05). Analysis of 35 serum and liver DNA samples from patients with hepatocellular carcinoma demonstrated that the amount of serum cccDNA is beyond the minimum detection limit and that the liver cccDNA quantity is about 0-49.2 copies/cell, consistent with previous reports. Taken together, this method has the potential for evaluating the efficacy of antiviral drugs. (C) 2010 Elsevier B.V. All rights reserved.
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