A nonradioactive riboprobe assay for the detection of hepatitis B virus DNA in human sera

Sequences of hepatitis B virus DNA were detected specifically in the sera of infected individuals by a non-radioactive riboprobe nucleic acid hybridization assay. The nucleic acids contained in serum specimens were prepared by an overnight proteinase K-/SDS-digest, phenol/chloroform-extraction and ethanol-precipitation, and immobilized on nylon membranes by the dot-blot technique. Digoxigenin-labelled 1.4 kb RNA-probes were generated by the phage sp 6 RNA-polymerase from a linearized HBV DNA transcription template containing the appropriate promoter. Hybridized probes were detected by alkaline phosphatase-conjugated sheep anti-digoxigenin Fab-fragments, and 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitroblue tetrazolium salt (NBT) as chromogenic substrates for the subsequent ELISA procedure. With a detection limit of 0.5-1.0 pg HBV DNA and a high specificity, the results were comparable to those obtained by the standard assay employing radioactively labeled DNA-probes.