Quantitation of hepatitis B virus DNA (HBV DNA) in serum using the spot hybridization technique and scintillation counting.
The simple spot hybridization technique to detect HBV DNA in serum was modified to concentrate Dane particles by pelleting. This minimised interference by serum components and allowed larger volumes of sera (up to 500 microliters) to be used in serial dilution on borderline positive samples and increased the efficiency of filtering through a "Hybri.Dot' system. Quantification of HBV DNA by 32P-scintillation Cerenkov counting based on serial standards of cloned HBV DNA processed through the "Hybri.dot' was easy to perform and the assay had good sensitivity (detection limit 2 pg, mode 6.25 pg), precision and accuracy. The pellet-simple spot quantitative assay proved more sensitive than those involving lengthier extraction procedures or direct application of serum to hybridization filters. Scintillation counting curves were linear over a wider range (0-800 pg HBV DNA) than optical densitometry measurements (0-50 pg) of autoradiographs. There was an excellent correlation with DNA polymerase activity (r = 0.948; P less than 0.001) but the assay proved more sensitive since HBV DNA (greater than 45 pg cloned equivalents) was detected in 4 out of 14 DNA polymerase negative sera and in 7 of 7 borderline (DNA polymerase cpm range 116-303) samples. This sensitive, quantitative HBV DNA assay should be of considerable value in studies on infectivity and effectiveness of antiviral therapies.
D004279:DNA, Viral / Q000097:blood*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003720:Densitometry; D006513:Hepatitis B e Antigens / Q000032:analysis; D006515:Hepatitis B virus / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D009693:Nucleic Acid Hybridization; D012588:Scintillation Counting
D004279:DNA, Viral; D006513:Hepatitis B e Antigens; D004259:DNA-Directed DNA Polymerase