Fully automated, internally controlled quantification of hepatitis B Virus DNA by real-time PCR by use of the MagNA Pure LC and LightCycler instruments.
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.
Leb
Victoria
V
Institute of Laboratory Medicine, General Hospital Linz, A-4020 Linz, Graz, Austria.
Stöcher
Markus
M
Valentine-Thon
Elizabeth
E
Hölzl
Gabriele
G
Kessler
Harald
H
Stekel
Herbert
H
Berg
Jörg
J
D001331:Automation / Q000295:instrumentation*; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000097:blood / Q000235:genetics*; D006509:Hepatitis B / Q000097:blood / Q000175:diagnosis*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods
D017931:DNA Primers; D004279:DNA, Viral