FLOW CYTOMETRIC DETECTION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROVIRAL DNA BY THE POLYMERASE CHAIN-REACTION INCORPORATING DIGOXIGENIN-LABELED OR FLUORESCEIN-LABELED DUTP

Serological assays are routinely used in the laboratory diagnosis of h uman immunodeficiency virus type-1 (HIV-1) infection, but the polymera se chain reaction (PCR) is ultimately the most sensitive and direct me thod 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. U sing heminested PCR we directly incorporated fluorescein-12-dUTP (fluo -dUTP) or digoxigenin-11-dUTP (dig-dUTP) into the PCR-amplicons. The l abeled amplicons were hybridized with biotinylated antisense and sense probes, fol lowed by capture of the hybrid DNA using streptavidin-coa ted beads which were finally analyzed in a now cytometer by 1) direct detection of the fluorescence intensity of the amplicons incorporating fluo-dUTP and 2) immunodetection of the amplicons incorporating dig-d UTP by anti-digoxigenin IgG labeled with fluorescein isothiocyanate (F ITC). Although both assays were functionally comparable with radiolabe led probe in reliably detecting as low as five copies of HIV-1 provira l DNA sequences, the immunodetection of dig-dUTP consistently yielded higher mean channel fluorescence and gave a stable signal over an exte nded period of 12-14 weeks, In testing a panel of 20 pedigreed PBMC sp ecimens from blood donors with or without HIV-1 infection, the results of both flow cytometric assays were identical with those of the conve ntional radioactive procedure. Therefore, we conclude that the dig-dUT P incorporation in amplicons, hybridization with a pair of sense-antis ense biotinylated probes and immunodetection of hybrids by flow cytome tric analyses is the nonisotopic method of choice for PCR-diagnosis of HIV-1 infection. (C) 1995 Wiley-Liss, Inc.