Real-time polymerase chain reaction assay for cell-associated HTLV type I DNA viral load

We have developed a quantitative real-time PCR assay for HTLV-I DNA, This a ssay approach uses real-time monitoring of fluorescent signal generation as a consequence of Tag-mediated amplification of specific target sequences t o allow real-time kinetic analysis of amplicon production. This kinetic app roach yields excellent sensitivity and an extremely broad linear dynamic ra nge, and ensures that quantitation is based on analysis during the exponent ial phase of amplification, regardless of the input template copy number. T he HTLV-I DNA assay has a nominal threshold sensitivity of 10 copy Eq/react ion, although single-copy plasmid template can be detected at frequencies c onsistent with statistical prediction. The linear dynamic range is in exces s of 5 logs, Interassay reproducibility averages 14% (coefficient of variat ion) for control templates over a range of 10(1) to 10(6) copy Eq/reaction and 25%, based on studies of extraction and analysis of replicate aliquots of PBMC specimens from HTLV-I-infected subjects. The primer/probe combinati on targets tax sequences conserved across described HTLV-I and HTLV-II isol ates. Parallel quantitation in the same samples of an endogenous sequence p resent at a known copy number per cell allows normalization of results for potential variation in DNA recovery. Availability of this assay should faci litate studies of basic pathogenesis and clinical evaluation of HTLV-I and HTLV-II infection, as well as assessment of therapeutic approaches.