Detection of HBV DNA using real time analysis.

Abstract

Isolation of HBV DNA was performed using the MagnaPure LC isolation station (Roche Applied Science, Penzberg, Germany), with a modified protocol HBV-02 to perform a proteinase K digestion initially. DNA was concentrated fourfold. Amplification was performed in a 50 ul reaction mixture, containing 20 ul isolated DNA, 2× TaqMan universal MasterMix (Applied BioSystems). After an incubation step of 2 min at 50 °C to inactivate possible contaminating amplicons using uracyl N -glycosylase, an incubation of 10 min at 95 °C activated AmpliTaq Gold polymerase. The PCR cycling program consists of 50 two step cycles of 15 s at 95 °C and 60 s at 60 °C. All reactions were performed in an 7700 SDS system (Applied BioSystems). * Corresponding author. Tel.: +31-10-463-3431; fax: +3110-463-3441 E-mail address: niesters@viro.fgg.eur.nl (H.G.M. Niesters).