Use of digoxigenin-labelled probes for the quantitation of HBV-DNA in antiviral drug evaluation.
The use of digoxigenin-labelled probes was studied for quantitation of HBV-DNA during antiviral drug evaluation. Digoxigenin (dig)-labelled probes were generated either via incorporation of dig-dUTP in a polymerase chain reaction (PCR) or a random priming reaction. Using the PCR-labelled probe (delineating a 523 bp fragment in the core gene of the HBV) as little as 1 pg of immobilized HBV-DNA could be detected following an 8 h exposure of the hybridized membrane. A close correlation (r = 0.95) was found between the amount of HBV-DNA (range 2.5-200 pg) and the signal generated by the probe hybridized to its target DNA. By using a probe that was labelled with digoxigenin via random priming, the minimal quantity of immobilized HBV plasmid DNA that could be detected following an 8 h exposure was 4 pg, whereas a 32P-labelled probe, generated in parallel by random priming, allowed the detection of 16 pg of HBV plasmid DNA following a 4-day exposure. The PCR-generated digoxigenin-labelled probe proved to be useful for antiviral drug evaluation, i.e. to detect HBV-DNA in total cellular DNA from HBV-positive hepatoma cells (HepG2.2.15) that had either been treated with reference antiviral agents or left untreated. The 50% effective concentrations (EC50) that were calculated for inhibition of HBV-DNA production by lamivudine (3TC), penciclovir (PCV), lobucavir (LBV), adefovir (PMEA) and tenofovir (PMPA) were comparable to those reported in the literature. The use of digoxigenin-labelled probes thus appears to be a simple, convenient, rapid, reliable and non-radioactive method for use for anti-HBV screening. In addition, and in contrast to 32P-labelled probes, digoxigenin-labelled probes can be stored for >1 year without loss of specific activity, which makes these probes particularly attractive for large-scale antiviral drug evaluation purposes.
Ying
C
C
Rega Institute for Medical Research, Leuven, Belgium.
Van Pelt
J
J
Yap
S H
SH
De Clercq
E
E
Neyts
J
J
D000998:Antiviral Agents / Q000494:pharmacology*; D015342:DNA Probes / Q000187:drug effects / Q000378:metabolism*; D004279:DNA, Viral / Q000032:analysis*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004076:Digoxigenin / Q000378:metabolism*; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D000998:Antiviral Agents; D015342:DNA Probes; D004279:DNA, Viral; D003856:Deoxyuracil Nucleotides; C027078:deoxyuridine triphosphate; D004076:Digoxigenin