ＨＴＬＶ‐Ｉ検出におけるＴａｑＭａｎ ＰＣＲ法の評価

Abstract

We used a novel “real time” quantitative PCR method, TagMan PCR (T-PCR), in which the accumulation of amplified products is detected via the formation of dual fluorogenic probe complexes, to diagnose HTLV-I infection, and compared the results with the Nested PCR method (N-PCR). Currently, confirmation of HTLV-I infection requires the detection of anti-HTLV-I by a serological method and of HTLV-I genome by a polymerase chain reaction (PCR)—based methodology. Secondary PCR-based assay is required to screen false positives which may occur during the serological testing. N-PCR is normally employed to amplify specific sequences from a very few copies of HTLV-I genome, this, however, consists of two PCR (time consuming) steps and is not intended for quantitative analysis. In our testing model as few as 3 copies of a partial genome of HTLV-I (3′ end half of HTLV-I genome cloned in plasmid) were successfully amplified and detected with comparable accuracy to N-PCR. Moreover, the measured quantitative values (Th cycle) of T-PCR were highly correlated with copy numbers of integrated HTLV-I genome estimated by other routine methodologies. Lastly, less labor was required to perform T-PCR than N-PCR, and the use of closed tubes in T-PCR significantly reduced carryover contamination. We conclude that T-PCR is a rapid and accurate assay system for confirmation of the serological diagnosis of HTLV-I infection.