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Journal of Clinical Microbiology, December 2001, p. 4339-4343, Vol. 39, No. 12
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.12.4339-4343.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Efficient Extraction of Virus DNA by NucliSens Extractor Allows Sensitive Detection of Hepatitis B Virus by PCR

Erik Gobbers,1 Tom A. M. Oosterlaken,1,* Mario J. A. W. M. van Bussel,2 Roel Melsert,1 Aloys C. M. Kroes,2 and Eric C. J. Claas2

Organon Teknika, Boxtel,1 and Department of Medical Microbiology, Leiden University Medical Center, Leiden,2 The Netherlands

Received 12 December 2001/Returned for modification 18 February 2001/Accepted 21 March 2001

The NucliSens Extractor is an automated nucleic acid isolation system based on guanidinium thiocyanate (GuSCN)-silica extraction technology. The system has been validated for the isolation of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNAs from human samples in combination with nucleic acid sequence-based amplification- and reverse transcription-PCR-based methods. We evaluated the extractor for hepatitis B virus (HBV) DNA extraction from human samples using a noncommercial HBV DNA PCR. Several sample pretreatment procedures in combination with the extractor were compared with the Qiagen extraction method, and the impact of the sample volume used in the extraction on the sensitivity was investigated. Heating of the lysed sample prior to extractor isolation and the use of a large sample volume resulted in highly sensitive detection of HBV DNA. Incubation of a 1-ml sample in GuSCN at 80°C (10 min) and at 37°C (30 min) allowed detection of 4 and 40 HBV genome equivalents/ml, respectively, in standard dilution panels. Sample lysis in GuSCN at room temperature and proteinase K treatment prior to use of the extractor were less efficient procedures. All clinical samples that were PCR positive after Qiagen extraction and/or that were HBsAg positive were also PCR positive after extractor isolation. HBV DNA, HCV RNA, and HIV type 1 RNA were efficiently coextracted from a single sample, allowing reliable detection of viral genomes.

* Corresponding author. Mailing address: Organon Teknika, Room F1202, Boseind 15, 5281 Boxtel, The Netherlands. Phone: 0031411 654635. Fax: 0031411654311. E-mail: t.oosterlaken{at}organonteknika.nl.

Journal of Clinical Microbiology, December 2001, p. 4339-4343, Vol. 39, No. 12
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.12.4339-4343.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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