Lightcycler 480 real time pcr 96 well system 2

HDV genomes were quantified as vGE using QuantiTect Virus Kit (Qiagen, Hilden, Germany) in a one-step qPCR. Reverse transcription was performed for 20 min at 50 °C followed by an initial denaturation step (5 min at 95 °C). Amplification occurred in 45 cycles of sequential denaturation (15 s at 95 °C) and primer annealing and extension (45 s 60 °C) steps. Analysis was performed in the LightCycler 480 Real-time PCR 96-well system II (Roche, Basel, Switzerland). Following oligonucleotides were used: CCC TTA GCC ATC CGA GTG G (HDV fw), TCC TCT TCG GGT CGG CA (HDV rev), ATG CCC AGG TCG GAC CGC G (HDV probe). The 1st WHO International Standard for HDV RNA, genotype 1 (Cat. NO. 7657/12, provided by PEI) was used for quantification.

HepG2 cells were seeded onto collagen-coated plates (6x10⁵ cells/well) and after 3 days of differentiation using 2.5% DMSO infected with HBV (MOI = 100) overnight. Infection was proven by establishment of nuclear cccDNA and HBeAg secretion. HBeAg was detected in the culture medium using a commercial immunoassay (Siemens Molecular Diagnostics, Marburg, Germany). Total DNA was extracted from infected cells 3 days post infection (PI) using a NucleoSpin tissue kit (Macherey Nagel, Düren, Germany) and subsequently treated with T5 exonuclease (10U/μl) for 10 min before cccDNA detection. HBV cccDNA was detected by real time PCR (qPCR) with selective PCR primers as previously described [27 (link), 28 (link)]. qPCR was performed on the LightCycler 480 real time PCR 96-well system II (Roche, 5015278001) and analysed using the second derivative maximum approach that takes normalisation to a reference gene (PrP) and primer efficiency into account.