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Journal of Clinical Microbiology, February 2004, p. 585-590, Vol. 42, No. 2
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.2.585-590.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Fully Automated, Internally Controlled Quantification of Hepatitis B Virus DNA by Real-Time PCR by Use of the MagNA Pure LC and LightCycler Instruments

Victoria Leb,1 Markus Stöcher,1 Elizabeth Valentine-Thon,2 Gabriele Hölzl,1 Harald Kessler,3 Herbert Stekel,1 and Jörg Berg1*

Institute of Laboratory Medicine, General Hospital Linz, A-4020 Linz,1 Molecular Diagnostics Laboratory, Institute of Hygiene, Karl-Franzens-University Graz, A-8010 Graz, Austria,3 Laboratory Dr. M. Sandkamp, B. Köster, Dr. R. Hiller, 28259 Bremen, Germany2

Received 28 April 2003/ Returned for modification 14 July 2003/ Accepted 22 October 2003

We report on the development of a fully automated real-time PCR assay for the quantitative detection of hepatitis B virus (HBV) DNA in plasma with EDTA (EDTA plasma). The MagNA Pure LC instrument was used for automated DNA purification and automated preparation of PCR mixtures. Real-time PCR was performed on the LightCycler instrument. An internal amplification control was devised as a PCR competitor and was introduced into the assay at the stage of DNA purification to permit monitoring for sample adequacy. The detection limit of the assay was found to be 200 HBV DNA copies/ml, with a linear dynamic range of 8 orders of magnitude. When samples from the European Union Quality Control Concerted Action HBV Proficiency Panel 1999 were examined, the results were found to be in acceptable agreement with the HBV DNA concentrations of the panel members. In a clinical laboratory evaluation of 123 EDTA plasma samples, a significant correlation was found with the results obtained by the Roche HBV Monitor test on the Cobas Amplicor analyzer within the dynamic range of that system. In conclusion, the newly developed assay has a markedly reduced hands-on time, permits monitoring for sample adequacy, and is suitable for the quantitative detection of HBV DNA in plasma in a routine clinical laboratory.

* Corresponding author. Mailing address: Institute of Laboratory Medicine, General Hospital Linz, Krankenhausstrasse 9, A-4020 Linz, Austria. Phone: 0043-70-7806-1174. Fax: 0043-70-7806-1815. E-mail: joerg.berg{at}akh.linz.at.

Journal of Clinical Microbiology, February 2004, p. 585-590, Vol. 42, No. 2
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.2.585-590.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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