Detection of HBV DNA in HBsAg-positive sera after amplification using the polymerase chain reaction.
The presence of hepatitis B virus (HBV) DNA in serum as detected by molecular hybridization is considered the most reliable marker for the presence of complete virions and, therefore, infectivity. This technique, however, has a lower limit of detection of 0.1 pg HBV DNA. Using the polymerase chain reaction (PCR), a technique by which DNA sequences can be amplified selectively, we investigated sera from 30 HBsAg carriers, 6 also positive for HBeAg and 24 negative for HBeAg. After PCR followed by Southern blot, 27 sera were found to be positive for HBV DNA, whereas only 7 sera were positive for HBV DNA in the conventional dot blot. PCR followed by Southern blot analysis lowered the limit of detection to 0.5 fg HBV DNA. Amplified HBV DNA fragments from some samples were directly sequenced without previous cloning. We conclude that PCR is a suitable method to amplify parts of viral genomes present in human sera, and that PCR with subsequent Southern blot analysis allows the detection of hepatitis B virions in the majority of HBsAg-positive sera.
Theilmann
L
L
Department of Internal Medicine, University of Heidelberg, Federal Republic of Germany.
Fischer
M
M
Galle
P R
PR
Nassal
M
M
D015139:Blotting, Southern; D004279:DNA, Viral / Q000097:blood*; D006509:Hepatitis B / Q000382:microbiology*; D006514:Hepatitis B Surface Antigens / Q000032:analysis*; D006513:Hepatitis B e Antigens / Q000032:analysis; D006515:Hepatitis B virus / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction; D014771:Virion / Q000302:isolation & purification
D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens