QUANTIFICATION OF HEPATITIS-B VIRUS-DNA BY COMPETITIVE AMPLIFICATION AND HYBRIDIZATION ON MICROPLATES

Present methods for quantification of hepatitis B virus (HBV) particle s from serum samples are not sensitive enough for some recent clinical applications. We describe a test that allows quantification of HBV DN A in a broad dynamic range from less than 40 to 10(6) molecules based on competitive PCR. The specimen DNA and a known amount of an internal standard (IS) are co-amplified in the same tube with the same primers , one of which is biotinylated. The two biotinylated products can be q uantified by hybridization on microplates coated with streptavidin, be cause their internal sequences are nonhomologous. An adequate standard curve is obtained by amplifying HBV DNA from a plasmid clone together with an IS. The ratio of amplified HBV DNA to IS DNA enables quantifi cation of the original amount of HBV without tedious titrations of eac h sample with competitor. The lower limit for quantitative analysis wi th radioactive probes was between 4 and 40 virus particles in a 10-mul serum sample.