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Journal of Clinical Microbiology, September 1999, p. 2899-2903, Vol. 37, No. 9
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Quantitation of Hepatitis B Virus Genomic DNA by Real-Time Detection PCR

Aki Abe,1 Kazuaki Inoue,2,3,* Takeshi Tanaka,4 Junko Kato,2,5 Naoki Kajiyama,1 Ryuji Kawaguchi,1 Satoshi Tanaka,4 Makoto Yoshiba,3 and Michinori Kohara2

Center for Molecular Biology and Cyotogenesis, SRL, Inc., Hino Tokyo 192-0002,1 Department of Microbiology, The Tokyo Metropolitan Institute of Medical Science,2 and Liver Unit, The Tokyo Metropolitan Komagome Hospital,4 Bunkyo-ku, Tokyo 113-8613, Division of Gastroenterology, Showa University Fujigaoka Hospital, Aoba-ku, Yokohama 227-8501,3 and Institute of Gastroenterology, Tokyo Women's Medical College, Shinjiyuku-ku, Tokyo 162-0054,5 Japan

Received 28 December 1998/Returned for modification 3 March 1999/Accepted 15 June 1999

Quantitation of hepatitis B virus (HBV) DNA in serum is a useful method for the monitoring of HBV replication. We attempted to develop a quantitative assay system for HBV DNA that is more sensitive, accurate, and reproducible than existing systems. We detected HBV DNA by real-time detection PCR (RTD-PCR) based on Taq Man chemistry. The efficacy of this assay was evaluated by quantitatively measuring sequential levels of synthetic DNA and DNA in clinical serum samples. The detection limit of this system was as few as 10 DNA copies/reaction. A linear standard curve was obtained between 101 and 108 DNA copies/reaction. The coefficient of variation for both intra- and interexperimental variability indicated remarkable reproducibility. This system detected HBV DNA in 100% of chronic hepatitis B patients tested and never detected HBV DNA in healthy volunteers who were negative for HBV markers. These observations suggest that RTD-PCR is an excellent candidate for a standard HBV quantification method.

* Corresponding author. Mailing address: Department of Microbiology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. Phone: 81-3-3823-2101. Fax: 81-3-3828-8945. E-mail: kinoue{at}rinshoken.or.jp.

Journal of Clinical Microbiology, September 1999, p. 2899-2903, Vol. 37, No. 9
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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