QUANTITATIVE PCR FOR HEPATITIS-B VIRUS WITH COLORIMETRIC DETECTION

A novel, sensitive colorimetric test is described for quantification o f the initial number of hepatitis B virus (HBV) genomes amplified in P CR. The viral genomes are amplified together with a synthetic internal standard (IS) to correct for the variability of the efficiency factor . One of the two primers is biotinylated, and the amplified mixtures o f HBV and IS DNAs are bound to streptavidin-coated microtiter plates f or quantitative detection. The ratio of HBV to IS DNA is determined fo r each sample by hybridization with DNP-containing probes and immunoen zymatic detection. The colorimetric detection is quantitative, rapid, and accurate with a dynamic range from similar to 10(8) to >10(11) DNA molecules. The initial number of HBV genomes in a clinical sample is interpreted from the signal ratio HBV/IS by using a standard curve, ob tained from coamplification of known quantities of synthetic HBV templ ates with IS. The assay quantified 15 viral genomes from 10 mu l of se rum, and its dynamic range was up to five orders of magnitude. After t he amplification step, the assay takes <2 hr, and the method is applic able to automation.