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Detection of hepatitis B virus in plasma using flow cytometric analyses of
polymerase chain reaction-amplified DNA incorporating digoxigenin-11- dUTP
G Yang, PP Ulrich, RA Aiyer, BD Rawal and GN Vyas
Department of Laboratory Medicine, University of California, School of
Medicine, San Francisco 94143-0134.
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.
Volume 81,
Issue 4,
pp. 1083-1088,
02/15/1993
Copyright © 1993 by The American Society of Hematology
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