Abstract
Present methods for quantification of hepatitis B virus (HBV) particles
from serum samples are not sensitive enough for some recent clinical
applications. We describe a test that allows quantification of HBV DNA
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
quantified by hybridization on microplates coated with streptavidin,
because 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
quantification of the original amount of HBV without tedious titrations
of each sample with competitor. The lower limit for quantitative
analysis with radioactive probes was between 4 and 40 virus particles in
a 10-microliters serum samples.
Original language | English |
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Pages (from-to) | 134-139 |
Journal | BioTechniques |
Volume | 15 |
Issue number | 1 |
Publication status | Published - 1993 |
MoE publication type | A1 Journal article-refereed |