Detection of hepatitis B virus in plasma using flow cytometric analyses of polymerase chain reaction-amplified DNA incorporating digoxigenin-11-dUTP.
Blood donations are routinely screened by multiple serologic assays for antigens/antibodies associated with infection by blood-borne viruses, including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency viruses (HIV-1 and HIV-2), and human T-cell lymphotropic virus (HTLV-I and HTLV-II). A direct detection of these viruses would be more effective for the prevention of transfusion-transmitted infections than the indirect measurement of the variable host immune response to these agents. Because the polymerase chain reaction (PCR) for viral gene amplification offers the most sensitive and direct means of detecting viruses in blood, we have developed a nonisotopic PCR procedure for the detection of HBV, chosen as a prototype. The problems, common to previously described PCR methods, of nucleic acid extraction and inhibition of the PCR by plasma proteins were overcome by isolation of HBV from plasma by means of 450-microns polystyrene beads covalently coated with monoclonal antibody to the Pre-S1 region of the viral envelope protein. Detergent lysis and proteinase K digestion of the immunocaptured virions isolated from plasma released the HBV DNA. A modified PCR-amplification protocol, incorporating digoxigenin-labeled dUTP in the amplified gene products followed by hybridization with a specific biotinylated oligonucleotide probe bound to streptavidin-coated 2.8-microns magnetic beads, allowed flow cytometric analyses of HBV-specific PCR products by means of antibodies to digoxigenin labeled with fluorescein isothiocyanate. The endpoint serial dilutions of pedigreed human plasma samples containing chimpanzee infectious dose (CID50) of 10(7) for adw and CID50 of 10(7.5) for the ayw subtypes were compared in repeated testing of PCR products by our immunoreactive bead (PCR-IRB) assay. HBV DNA was consistently detected in a 5 x 10(-10) dilution of each sample. In testing 20 coded specimens of blood donors, with or without serologic markers of HBV infection, the PCR-IRB was specific and more sensitive than the PCR analyses by slot blot hybridization with radioactive probe. The PCR-IRB assay can be adapted for simultaneous detection of multiple blood-borne viruses by an automated flow cytometric analysis system.
Yang
G
G
Department of Laboratory Medicine, University of California, School of Medicine, San Francisco 94143-0134.
Ulrich
P P
PP
Aiyer
R A
RA
Rawal
B D
BD
Vyas
G N
GN
D001426:Bacterial Proteins; D004279:DNA, Viral / Q000097:blood*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D003902:Detergents; D004076:Digoxigenin / Q000031:analogs & derivatives* / Q000378:metabolism; D019286:Endopeptidase K; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006509:Hepatitis B / Q000382:microbiology*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008280:Magnetics; D008863:Microspheres; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012697:Serine Endopeptidases; D019809:Streptavidin
D001426:Bacterial Proteins; D004279:DNA, Viral; D003856:Deoxyuracil Nucleotides; D003902:Detergents; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; C070202:digoxigenin-11-deoxyuridine triphosphate; D019809:Streptavidin; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D016650:Fluorescein-5-isothiocyanate; D004076:Digoxigenin