Quantitative assay of PCR-amplified hepatitis B virus DNA using a peroxidase-labelled DNA probe and enhanced chemiluminescence.
We have developed a sensitive and quantitative assay for hepatitis B virus (HBV) DNA in serum or plasma in which PCR and then microtiter hybridization analysis are used. Assay of HBV DNA in serum or plasma is important for demonstrating viral replication, indicating and monitoring antiviral therapy, determining the infectivities of virus carriers, and ensuring the safety of blood products. Under optimum conditions PCR can amplify one HBV DNA molecule to 10(8) copies, but detection of this amount of DNA still requires hybridization with labelled probes or a nested PCR. We labelled one strand of the PCR product with a biotinylated primer. The double-stranded amplicon was incubated in streptavidin-coated microplate wells. The nonlabelled strand was removed after denaturation of the double-stranded DNA with alkali, and the bound strand was hybridized with a peroxidase-coupled single-stranded probe. The amount of bound peroxidase was measured in a luminometer. Four picograms of amplicon was detectable in this system, whereas conventional ethidium bromide staining requires a 1,000 times higher amplicon concentration. The performance of the new assay was compared with those of nested PCR and a PCR system that uses a digoxigenin label, hybridization to a solid-phase adsorbed probe, and colorimetric detection. The chemiluminescence assay was found to be almost as sensitive as nested PCR and approximately five times more sensitive than the colorimetric test.
Erhardt
A
A
Institute of Medical Virology, Justus-Liebig-University of Giessen, Germany.
Schaefer
S
S
Athanassiou
N
N
Kann
M
M
Gerlich
W H
WH
D001426:Bacterial Proteins; D001710:Biotin; D015342:DNA Probes; D004279:DNA, Viral / Q000097:blood*; D006509:Hepatitis B / Q000097:blood*; D006801:Humans; D008163:Luminescent Measurements; D015336:Molecular Probe Techniques; D009195:Peroxidase; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019809:Streptavidin
D001426:Bacterial Proteins; D015342:DNA Probes; D004279:DNA, Viral; D001710:Biotin; D019809:Streptavidin; D009195:Peroxidase