Typing human T-cell lymphotropic virus (HTLV-I and HTLV-II) by nested polymerase chain reaction: application to clinical specimens.
Human T-cell lymphotropic virus type I and II provirus DNA was detected 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 consists of first amplification which detects both HTLV-I and HTLV-II, and a second amplification (nested-PCR) to discriminate between the two viruses and to improve sensitivity. Optimal conditions of MgCl2 concentration and annealing temperature were found for maximal amplification and specificity. This method was used for the amplification of conserved regions of pol and env genes. 1.5 pg of MT-2 and 5 pg of C3/44 Mo cell 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 without the use of type-specific radiolabeled probes. Furthermore, this method can detect and discriminate the two viruses in one step PCR using the primers used in the nested-PCR. Nevertheless, there is a decrease in sensitivity of 100-fold. The results of five seropositive samples confirmed by Western blot are compared with PCR. PCR typed one of these samples as HTLV-I and the rest as HTLV-II. This technique is useful in cases such as window period, perinatal studies and when serologic results are not satisfactory.
Vallejo
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Servicio de Diagnóstico y Referencia, Instituto de Salud Carlos III, Madrid, Spain.
García-Sáiz
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D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005069:Evaluation Studies as Topic; D015752:Genes, env; D015751:Genes, pol; D015490:HTLV-I Infections / Q000175:diagnosis / Q000821:virology; D015491:HTLV-II Infections / Q000175:diagnosis / Q000821:virology; D015368:Human T-lymphotropic virus 1 / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D015367:Human T-lymphotropic virus 2 / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D011533:Proviruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods / Q000706:statistics & numerical data
D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral