       Document 0433
 DOCN  M9630433
 TI    Flow cytometric immunodetection of human immunodeficiency virus type 1
       proviral DNA by heminested PCR and digoxigenin-labeled probes.
 DT    9603
 AU    Yang G; Garhwal S; Olson JC; Vyas GN; Department of Laboratory Medicine,
       University of California, San; Francisco 94143-0134, USA.
 SO    Clin Diagn Lab Immunol. 1994 Jan;1(1):26-31. Unique Identifier :
       AIDSLINE MED/96050774
 AB    PCR is the most sensitive and direct method for detecting blood-borne
       viruses, as well as an efficient means for producing vector-free probes.
       However, the application of PCR, especially in the laboratory diagnosis
       of human immunodeficiency virus (HIV) infection, is impeded by the
       current use of radiolabeled oligonucleotide probes. Therefore, we have
       developed a nonisotopic PCR immunoreactive bead (PCR-IRB) assay to
       detect HIV type 1 proviral DNA from peripheral blood mononuclear cells
       (PBMC). We used a biotinylated primer in a set of three oligonucleotides
       selected from the HIV long terminal repeat region for heminested PCR
       amplification. An internal probe was synthesized by PCR with
       incorporation of digoxigenin-labeled dUTP. After solution hybridization
       of the probe with PCR-amplified products (amplicons), the hybridized DNA
       was captured with streptavidin-coated magnetic beads. For the detection
       of hybrids, flow cytometric analyses were carried out by two procedures:
       (i) direct detection with fluorescein isothiocyanate (FITC)-labeled
       antidigoxigenin immunoglobulin G (IgG) antibody and (ii) indirect
       detection with antidigoxigenin sheep IgG antibody followed by
       FITC-labeled anti-sheep IgG antibody. Both procedures in the PCR-IRB
       assay detected two to three copies of HIV proviral DNA sequences, a
       sensitivity that is comparable with that of the conventional radioactive
       detection of amplicons following probe hybridization and
       electrophoresis. To compare the PCR-IRB assay with the conventional
       method, we tested 53 pedigreed PBMC specimens from blood donors and
       newborns; the results obtained were identical. This nonisotopic PCR-IRB
       assay can also be automated for potential application in laboratory
       diagnosis of HIV infection, blood bank screening, and therapeutic
       monitoring of viremia and perinatal transmission.
 DE    Base Sequence  Comparative Study  Digoxigenin  DNA Primers  *DNA Probes
       DNA, Viral/*ISOLATION & PURIF  Female  *Flow Cytometry  Human
       HIV-1/*GENETICS/ISOLATION & PURIF  Immunomagnetic Separation  Infant,
       Newborn  Leukocytes, Mononuclear/VIROLOGY  Microspheres  Molecular
       Sequence Data  Polymerase Chain Reaction/*METHODS
       Proviruses/*GENETICS/ISOLATION & PURIF  Support, U.S. Gov't, P.H.S.
       JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

