title: Flow cytometric detection of human immunodeficiency virus type 1 proviral DNA by the polymerase chain reaction incorporating digoxigenin- or fluorescein-labeled dUTP.
abstract: 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.
authors.affiliation: Department of Laboratory Medicine, University of California, San Francisco 94143, USA.
authors.forename: G
authors.initials: G
authors.lastname: Yang
authors.forename: J C
authors.initials: JC
authors.lastname: Olson
authors.forename: R
authors.initials: R
authors.lastname: Pu
authors.forename: G N
authors.initials: GN
authors.lastname: Vyas
mesh_terms: D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis*; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015658:HIV Infections / Q000175:diagnosis*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016376:Oligonucleotides, Antisense; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification; D013942:Thymine Nucleotides
chemical_list: D004279:DNA, Viral; D003856:Deoxyuracil Nucleotides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D016376:Oligonucleotides, Antisense; D013942:Thymine Nucleotides; C027078:deoxyuridine triphosphate; D004076:Digoxigenin; C024157:thymidine 5'-triphosphate