QUANTITATIVE ASSAY OF PCR-AMPLIFIED HEPATITIS-B VIRUS-DNA USING A PEROXIDASE-LABELED DNA-PROBE AND ENHANCED CHEMILUMINESCENCE

We have developed a sensitive and quantitative assay for hepatitis B v irus (HBV) DNA in serum or plasma in which PCR and then microtiter hyb ridization analysis are used. Assay of HBV DNA in serum or plasma is i mportant for demonstrating viral replication, indicating and monitorin g antiviral therapy, determining the infectivities of virus carriers, and ensuring the safety of blood products. Under optimum conditions PC R can amplify one HBV DNA molecule to 10(8) copies, but detection of t his amount of DNA still requires hybridization with labelled probes or a nested PCR. We labelled one strand of the PCR product with a biotin ylated primer. The double-stranded amplicon was incubated in streptavi din-coated microplate wells. The nonlabelled strand was removed after denaturation of the double-stranded DNA with alkali, and the bound str and was hybridized with a peroxidase-coupled single-stranded probe, Th e amount of bound peroxidase was measured in a luminometer. Four picog rams of amplicon was detectable in this system, whereas conventional e thidium bromide staining requires a 1,000 times higher amplicon concen tration. The performance of the new assay was compared with those of n ested PCR and a PCR system that uses a digoxigenin label, hybridizatio n to a solid-phase adsorbed probe, and colorimetric detection. The che miluminescence assay was found to be almost as sensitive as nested PCR and approximately five times more sensitive than the colorimetric tes t.