Efficient extraction of virus DNA by NucliSens extractor allows sensitive detection of hepatitis B virus by PCR

The NucliSens Extractor is an automated nucleic acid isolation system based on guanidinium thiocyanate (GuSCN)-silica extraction technology. The syste m has been validated for the isolation of human immunodeficiency virus (HIV ) and hepatitis C virus (HCV) RNAs from human samples in combination with n ucleic acid sequence-based amplification- and reverse transcription-PCR-has ed methods. We evaluated the extractor for hepatitis B virus (HBV) DNA extr action from human samples using a noncommercial HBV DNA PCR. Several sample pretreatment procedures in combination with the extractor were compared wi th the Qiagen extraction method, and the impact of the sample volume used i n the extraction on the sensitivity was investigated. Heating of the lysed sample prior to extractor isolation and the use of a large sample volume re sulted in highly sensitive detection of HBV DNA. Incubation of a 1-ml sampl e in GuSCN at 80 degreesC (10 min) and at 37 degreesC (30 min) allowed dete ction of 4 and 40 HBV genome equivalents/ml, respectively, in standard dilu tion panels. Sample lysis in GuSCN at room temperature and proteinase K tre atment prior to use of the extractor were less efficient procedures. All cl inical samples that were PCR positive after Qiagen extraction and/or that w ere HBsAg positive were also PCR positive after extractor isolation. HBV DN A, HCV RNA, and HIV type 1 RNA were efficiently coextracted from a single s ample, allowing reliable detection of viral genomes.