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

Abstract

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 the major HBV subtypes were synthesized and used in an assay that could be completed in less than 6 h. The quantification range of the assay, as determined by testing serial dilutions of Eurohep HBV reference standards and cloned HBV DNA, was 5 x 10(5) to 3 x 10(9) molecules of HBV DNA/ml of serum. Within-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 after testing the same samples with the Chiron branched-DNA (bDNA) assay for HBV 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 samples, 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 had 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 required retesting by the cross-linking assay. The correlation between the HBV 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 reproducible method for the detection and quantification of HBV DNA levels in serum.

FIG. 1

Sensitivity of the cross-linking assay with different HBV subtypes. Eurohep reference samples 1 (subtype adw) and 2 (subtype ayw) were diluted in HBV-negative sera to give viral titers ranging from 0.1 to 100 Meq/ml of serum. The fluorescent signal obtained from the testing of each diluted sample was divided by the signal generated from control (HBV-negative) serum to give the signal/negative control ratio, as shown. Each point is the mean ratio obtained from tests with each sample performed in duplicate. The CVs between the duplicate samples at each point were <5%.

FIG. 2

Comparison of the HBV DNA levels in 194 serum samples determined by the cross-linking and bDNA assays. The line passing through the data with a slope equal to 1 is the hypothetical line that all datum points would fall on if the two assays yielded results in complete agreement with one another.