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Journal of Clinical Microbiology, August 2004, p. 3438-3440, Vol. 42, No. 8
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.8.3438-3440.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Real-Time PCR Assay Using Molecular Beacon for Quantitation of Hepatitis B Virus DNA

Simon Siu-Man Sum,1 Danny Ka-Ho Wong,1 Man-Fung Yuen,1 He-Jun Yuan,2 Jian Yu,3 Ching-Lung Lai,1* David Ho,3 and Linqi Zhang3*

Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong Special Administrative Region,1 Department of Medicine, Zhongshan Hospital, Fudan University, Shanghai, People's Republic of China,2 The Aaron Diamond AIDS Research Center, Rockefeller University, New York, New York3

Received 26 January 2004/ Returned for modification 17 March 2004/ Accepted 22 April 2004

Levels of hepatitis B virus (HBV) DNA in the blood serve as an important marker in monitoring the disease progression and treatment efficacy of chronic HBV infection. Several commercial assays are available for accurate measurement of HBV genomic DNA, but many of them are hampered by relatively low sensitivity and limited dynamic range. The aim of this study was to develop a sensitive and accurate assay for measuring HBV genomic DNA using real-time PCR with a molecular beacon (HBV beacon assay). The performance of this assay was validated by testing serial dilutions of the two EUROHEP HBV DNA standards (ad and ay subtypes) of known concentrations. The assay showed low intra-assay (<7%) and interassay (<5%) variations for both subtypes. Its dynamic range was found to be 101 to 107 copies per reaction (1.0 x 102 to 1.0 x 109 copies ml–1). The assay was further evaluated clinically using serum samples from 175 individuals with chronic hepatitis B. The HBV DNA level measured by this assay showed good correlation with that measured by the commercially available COBAS AMPLICOR HBV Monitor test (r = 0.901; P < 0.001). The higher sensitivity and broader dynamic range of this assay compared to the existing commercial assays will provide an ideal tool for monitoring disease progression and treatment efficacy in HBV-infected patients, in particular for those with low levels of HBV viremia.

* Corresponding author. Mailing address for Ching-Lung Lai: Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong. Phone: (852) 28554252. Fax: (852) 28162863. E-mail: hrmelcl{at}hkucc.hku.hk. Mailing address for Linqi Zhang: The Aaron Diamond AIDS Research Center, 455 First Ave., New York, NY 10016. Phone: (212) 448-5000. Fax: (212) 725-1126. E-mail: LZhang{at}adarc.org.

Journal of Clinical Microbiology, August 2004, p. 3438-3440, Vol. 42, No. 8
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.8.3438-3440.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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