Quantification of hepatitis B virus DNA by competitive amplification and hybridization on microplates

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....

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Bibliographic Details
Published in:	BioTechniques Vol. 15; no. 1; p. 134
Main Authors:	Jalava, T, Lehtovaara, P, Kallio, A, Ranki, M, Söderlund, H
Format:	Journal Article
Language:	English
Published:	England 01-07-1993
Subjects:	Bacterial Proteins Binding, Competitive Biotin Cloning, Molecular DNA, Viral - analysis Hepatitis B virus - genetics Hot Temperature Humans Nucleic Acid Denaturation Nucleic Acid Hybridization Polymerase Chain Reaction Streptavidin
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Summary:	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.
ISSN:	0736-6205