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A multiplex 'real-time' polymerase chain reaction (PCR) has been established as a general technique for the quantitation of proviral human T-lymphotrophic virus types 1 and 2 (HTLV-I/II). The technology utilizes fluorescence to measure amplification products from the tax gene of Human T-cell lymphotropic virus type 1 or the 5' long terminal repeat of Human T-cell lymphotropic virus type 2. The quantitative amplification of the standard was linear across four orders of magnitude with nearly identical amplification efficiencies for monoplex or the biplex format from 1.4 copes/assay (60 copies proviral DNA/0.5 micrograms human DNA) to 6000 copies/assay (240000 proviral copies/0.5 micrograms human DNA). The human beta-globin gene was used to normalize for human DNA input to determine the proviral DNA load. Three hundred fifty-six specimens received by Specialty Laboratories for HTLV I/II detection provided identical results in the detection of HTLV I/II proviral DNA. No additional positive specimens were identified with the biplex assay format. The coefficient of variation for the proviral DNA load was less than 30% for HTLV I or II quantitation (n=5). For spiked specimens, two groups of five separate 0.25 ml blood specimens (20 total) were spiked, respectively, with 0, 9.6, 48, 240 and 1200 copies of HTLV I or HTLV II DNA standards. The specimens were amplified with the HTLV I/II multiplex format. Twenty of twenty expected negative HTLV I or HTLV II specimens were negative (100% specificity) and 14/16 specimens spiked with 48 copies or more HTLV I were detected (87.5% sensitivity). Thirteen of sixteen HTLV II spiked specimens (>48 copies of HTLV II standard per 10 assays) were detected (81.2%). The real-time detection provides accurate and reliable results in a single amplification for both HTLV (I or II) targets with a more rapid turnaround time and a decrease in material required for results.