RAPID, SENSITIVE, SPECIFIC, AND QUANTITATIVE DETECTION OF HUMAN T-CELL LEUKEMIA-VIRUS TYPE-1 SEQUENCE IN PERIPHERAL-BLOOD MONONUCLEAR-CELLS BY AN IMPROVED POLYMERASE CHAIN-REACTION METHOD WITH NESTED PRIMERS

Improving on the nested double polymerase chain reaction (PCR) described previously, we have developed a new two-step PCR (TS-PCR) method for detecting more specifically the human T-cell leukemia virus type 1 (HTLV-1) proviral sequences in peripheral blood mononuclear cells (PBMC). In our TS-PCR method. the point of modification is to use optimal concentrations of primers in the first amplification step in the range of 0.01-0.025-mu-M. This increases sensitivity and specificity enough to detect from 1 to 10(5) copies of template DNA without radio-isotopes. This method is rapid because of completion in 1 day and is also applicable for quantitative detection of clinical specimens. The data show that the quantitative detection of HTLV-1 proviral sequences by this method correlates with the anti-HTLV-1 antibody titers from serologic analysis of seropositive healthy carriers. Moreover, the TS-PCR method using each specific primer was also attempted for successful detection of other viral genomes; therefore,the principle of this method is widely suitable for routine detection of genomes in the basic and clinical microbiological fields.