Detection of hepatitis B and woodchuck hepatitis viral DNA in plasma and mononuclear cells from heparinized blood by the polymerase chain reaction.
Amplification by the polymerase chain reaction (PCR) of hepatitis B virus (HBV) DNA extracted from parallel samples of serum and heparinized plasma gave contradictory results, indicating that heparin inhibits virus detection. Similarly, analysis of PCR products of woodchuck hepatitis virus (WHV) DNA showed that heparinization of blood abolished WHV DNA amplification, while anticoagulation with sodium EDTA or acid citrate dextrose did not. Amplification of recombinant WHV and HBV DNA in the presence of increasing concentrations of sodium heparin progressively inhibited and finally abolished virus genome detection. The inhibitory effect of heparin was reversed by treatment of either plasma or isolated DNA with heparinase (5 U/reaction, 1 h at 28 degrees C) prior to PCR. In contrast, heparin did not influence the detection of hepadnavirus in peripheral blood mononuclear cells (PBMC), even after prolonged incubation of the cells with heparin in culture. These findings confirm that heparin exerts a dramatic inhibitory effect on hepadnaviral DNA detection by PCR and they demonstrate that this effect can be reversed by heparinase. The findings also show that extensively washed PBMC derived from heparinized blood can be a reliable source of nucleic acids for amplification of hepadnavirus genome. These results imply that previous data should be reassessed if samples of heparinized plasma were found hepadnavirus DNA nonreactive by PCR or when these samples were used as a starting material for PCR quantitation of viral genome.
Pardoe
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Liver Research Laboratory, Faculty of Medicine, Health Sciences Complex, Memorial University of Newfoundland, St. John's, Canada.
Michalak
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D000818:Animals; D001483:Base Sequence; D019343:Citric Acid; D004279:DNA, Viral / Q000097:blood*; D004492:Edetic Acid; D005947:Glucose / Q000031:analogs & derivatives; D006493:Heparin; D019763:Heparin Lyase; D017919:Hepatitis B Virus, Woodchuck / Q000235:genetics / Q000302:isolation & purification*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear; D008392:Marmota; D008969:Molecular Sequence Data; D010949:Plasma; D016133:Polymerase Chain Reaction / Q000379:methods*; D011133:Polysaccharide-Lyases
D004279:DNA, Viral; C002113:acid citrate dextrose; D019343:Citric Acid; D006493:Heparin; D004492:Edetic Acid; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase; D005947:Glucose