title: Detection of amplified HTLV-I/-II viral sequences using time-resolved fluorometry.
abstract: Since its discovery, the polymerase chain reaction (PCR) has been used for different purposes in the field of DNA research. We tested the PCR for the diagnosis of HTLV-I/-II infections. PCR was used to amplify 141- and 149-base pair regions from the HTLV-I and HTLV-II virus genomes, respectively. The annealing temperature in the PCR amplification was optimized using 20% polyacrylamide gels and silver staining. Even a slight change (3 degrees C) in the annealing temperature had an effect on the specificity of the reaction. The PCR products were detected with biotin and Eu-labeled oligonucleotide probes in a solution hybridization format. The linearity of the assay was tested with serial dilutions of purified chromosomal DNA containing integrated HTLV-II sequences. The linearity was found to be dependent on the number of cycles used in the PCR amplification. The best linearity, at a target level of a few copies, was achieved using a low number of cycles. The specificity of the assay was tested using HTLV-I and HTLV-II-infected lymphocytes from the cell lines Hut102 and MO480, respectively. No cross reactivity between these analytes was observed.
authors.affiliation: Pharmacia Genetic Engineering, La Jolla, California 92037.
authors.forename: A
authors.initials: A
authors.lastname: Iitiä
authors.forename: P
authors.initials: P
authors.lastname: Dahlen
authors.forename: M
authors.initials: M
authors.lastname: Nunn
authors.forename: V M
authors.initials: VM
authors.lastname: Mukkala
authors.forename: H
authors.initials: H
authors.lastname: Siitari
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D015490:HTLV-I Infections / Q000175:diagnosis*; D015491:HTLV-II Infections / Q000175:diagnosis*; D015368:Human T-lymphotropic virus 1 / Q000235:genetics* / Q000302:isolation & purification; D015367:Human T-lymphotropic virus 2 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction
chemical_list: D004279:DNA, Viral