Nucleic acid-based cross-linking assay for detection and quantification ofhepatitis B virus DNA

A nucleic acid photo-cross-linking technology was used to develop a direct assay for the quantification of hepatitis B virus (HBV) DNA levels in serum . Cross-linker-modified DNA probes complementary to the viral genomes of th e major HBV subtypes were synthesized and used in an assay that could be co mpleted in less than 6 h, The quantification range of the assay, as determi ned by testing serial dilutions of Eurohep HBV reference standards and clon ed HBV DNA, was 5 x 10(5) to 3 x 10(9) molecules of HBV DNA/ml of serum. Wi thin-run and between-run coefficients of variation (CVs) for the assay were 4.3 and 4.0%, respectively, The assay was used to determine HBV DNA levels in 302 serum samples, and the results were compared to those obtained afte r testing the same samples with the Chiron branched-DNA (bDNA) assay for HB V DNA, Of the samples tested, 218 were positive for HBV DNA by both assays and 72 gave results below the cutoff for both assays. Of the remaining 12 s amples, 10 were positive for HBV DNA by the cross-linking assay only; the 2 other samples were positive by the bDNA assay only. Twenty-eight samples h ad to be retested by the bDNA assay (CV, >20% between the results obtained from the testing of each sample in duplicate), whereas only three samples r equired retesting by the cross-linking assay, The correlation between the H BV DNA levels, as measured by the two tests, was very high (r = 0.902; P = 0.01). We conclude that the cross-linking assay is a sensitive and reproduc ible method for the detection and quantification of HBV DNA levels in serum .