Detection of amplified HTLV-I/-II viral sequences using time-resolved fluorometry. |
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Authors: | A Iiti? P Dahlen M Nunn V M Mukkala H Siitari |
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Institution: | Pharmacia Genetic Engineering, La Jolla, California 92037. |
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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. |
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