Document 0863
 DOCN  M9650863
 TI    Flow cytometric detection of human immunodeficiency virus type 1
       proviral DNA by the polymerase chain reaction incorporating digoxigenin-
       or fluorescein-labeled dUTP.
 DT    9605
 AU    Yang G; Olson JC; Pu R; Vyas GN; Department of Laboratory Medicine,
       University of California, San; Francisco 94143, USA.
 SO    Cytometry. 1995 Oct 1;21(2):197-202. Unique Identifier : AIDSLINE
       MED/96120901
 AB    Serological assays are routinely used in the laboratory diagnosis of
       human immunodeficiency virus type-1 (HIV-1) infection, but the
       polymerase chain reaction (PCR) is ultimately the most sensitive and
       direct method for establishing definitive diagnosis. As an alternative
       to the conventional radioactive PCR procedure we have developed and
       evaluated a pair of rapid nonradioisotopic flow cytometric detection
       methods. Using heminested PCR we directly incorporated
       fluorescein-12-dUTP (fluo-dUTP) or digoxigenin-11-dUTP (dig-dUTP) into
       the PCR-amplicons. The labeled amplicons were hybridized with
       biotinylated antisense and sense probes, followed by capture of the
       hybrid DNA using streptavidin-coated beads which were finally analyzed
       in a flow cytometer by 1) direct detection of the fluorescence intensity
       of the amplicons incorporating fluo-dUTP and 2) immunodetection of the
       amplicons incorporating dig-dUTP by anti-digoxigenin IgG labeled with
       fluorescein isothiocyanate (FITC). Although both assays were
       functionally comparable with radiolabeled probe in reliably detecting as
       low as five copies of HIV-1 proviral DNA sequences, the immunodetection
       of dig-dUTP consistently yielded higher mean channel fluorescence and
       gave a stable signal over an extended period of 12-14 weeks. In testing
       a panel of 20 pedigreed PBMC specimens from blood donors with or without
       HIV-1 infection, the results of both flow cytometric assays were
       identical with those of the conventional radioactive procedure.
       Therefore, we conclude that the dig-dUTP incorporation in amplicons,
       hybridization with a pair of sense-antisense biotinylated probes and
       immunodetection of hybrids by flow cytometric analyses is the
       nonisotopic method of choice for PCR-diagnosis of HIV-1 infection.
 DE    Base Sequence  Comparative Study  Deoxyuracil Nucleotides  Digoxigenin
       DNA, Viral/*ANALYSIS  Flow Cytometry/*METHODS  Fluoresceins  Fluorescent
       Dyes  Human  HIV Infections/*DIAGNOSIS  HIV-1/GENETICS/*ISOLATION &
       PURIF  Molecular Sequence Data  Oligonucleotide Probes
       Oligonucleotides, Antisense  Polymerase Chain Reaction/*METHODS
       Proviruses/GENETICS/ISOLATION & PURIF  Support, U.S. Gov't, P.H.S.
       Thymine Nucleotides  JOURNAL ARTICLE

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