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Journal of Clinical Microbiology, October 2001, p. 3656-3665, Vol. 39, No. 10
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.10.3656-3665.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Quantitative Detection of Hepatitis B Virus DNA by Real-Time Nucleic Acid Sequence-Based Amplification with Molecular Beacon Detection

Sol Yates,1,dagger Maarten Penning,1,* Jaap Goudsmit,1,2 Inge Frantzen,3 Bert van de Weijer,3 Dianne van Strijp,3 and Bob van Gemen4

Department of Human Retrovirology1 and Amsterdam Institute of Viral Genomics,2 Academic Medical Center, and PrimaGen,4 Amsterdam, and Nucleic Acid Diagnostics Department, Organon Teknika, Boxtel,3 The Netherlands

Received 2 March 2001/Returned for modification 12 June 2001/Accepted 19 July 2001

We have developed a hepatitis B virus (HBV) DNA detection and quantification system based on amplification with nucleic acid sequence-based amplification (NASBA) technology and real-time detection with molecular beacon technology. NASBA is normally applied to amplify single-stranded target RNA, producing RNA amplicons. In this work we show that with modifications like primer design, sample extraction method, and template denaturation, the NASBA technique can be made suitable for DNA target amplification resulting in RNA amplicons. A major advantage of our assay is the one-tube, isothermal nature of the method, which allows high-throughput applications for nucleic acid detection. The homogeneous real-time detection allows a closed-tube format of the assay, avoiding any postamplification handling of amplified material and therefore minimizing the risk of contamination of subsequent reactions. The assay has a detection range of 103 to 109 HBV DNA copies/ml of plasma or serum (6 logs), with good reproducibility and precision. Compared with other HBV DNA assays, our assay provides good sensitivity, a wide dynamic range, and high-throughput applicability, making it a viable alternative to those based on other amplification or detection methods.

* Corresponding author. Mailing address: Department of Human Retrovirology, Meibergdreef 59, Academic Medical Center, 1105 BA Amsterdam, The Netherlands. Phone: 31 (0)205667669. Fax: 31 (0)205669081. E-mail: m.penning{at}amc.uva.nl.

dagger Present address: Notox, Hertogenbosch, The Netherlands.

Journal of Clinical Microbiology, October 2001, p. 3656-3665, Vol. 39, No. 10
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.10.3656-3665.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.