Detection of amplified HTLV-I/-II viral sequences using time-resolved fluorometry.
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.
Iitiä
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Pharmacia Genetic Engineering, La Jolla, California 92037.
Dahlen
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P
Nunn
M
M
Mukkala
V M
VM
Siitari
H
H
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
D004279:DNA, Viral