TYPING HUMAN T-CELL LYMPHOTROPIC VIRUS (HTLV-I AND HTLV-II) BY NESTEDPOLYMERASE CHAIN-REACTION - APPLICATION TO CLINICAL SPECIMENS

Human T-cell lymphotropic virus type I and II provirus DNA was detecte d by polymerase chain reaction (PCR). MT-2 (HTLV-I infected), C3/44 Mo (HTLV-II infected) cell lines and peripheral blood mononuclear cells (PBMNC) from HTLV seropositive samples were used. The procedure consis ts of first amplification which detects both HTLV-I and HTLV-II, and a second amplification (nested-PCR) to discriminate between the two vir uses and to improve sensitivity. Optimal conditions of MgCl2 concentra tion and annealing temperature were found for maximal amplification an d specificity. This method was used for the amplification of conserved regions of pol and enu genes. 1.5 pg of MT-2 and 5 pg of C3/44 Mo cel l line DNAs were detected using nested-PCR and liquid hybridization in the pol system. The env system could detect 1.5 pg of MT-2 and 1.5 pg of C3/44 Mo cell lines DNAs using nested-PCR and liquid hybridization . The pol system can type both HTLV-I and HTLV-II in only two steps wi thout the use of type-specific radiolabeled probes. Furthermore, this method can detect and discriminate the two viruses in one step PCR usi ng the primers used in the nested-PCR. Nevertheless, there is a decrea se in sensitivity of 100-fold. The results of five seropositive sample s confirmed by Western blot are compared with PCR. PCR typed one of th ese samples as HTLV-I and the rest as HTLV-II. This technique is usefu l in cases such as window period, perinatal studies and when serologic results are not satisfactory.