A non-radioisotopic reverse transcriptase assay using biotin-11-deoxyuridinetriphosphate on primer-immobilized microtiter plates
| Autor: | Masaru Otani, David T. Imagawa, Junzo Mizoguchi, Masashi Tanno, Takeshi Urabe, Tomohiko Takasaki, Masuyo Nakai, Moon H. Lee, Kouichi Sano, Hidenari Kusakabe |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
Lysis
viruses Biotin Biology Sensitivity and Specificity Virus Microtiter plate chemistry.chemical_compound Virology chemistry.chemical_classification Avian Myeloblastosis Virus Avian Leukosis Virus RNA-Directed DNA Polymerase Templates Genetic Molecular biology Reverse transcriptase Retroviridae Enzyme Oligodeoxyribonucleotides chemistry HIV-2 HIV-1 Primer (molecular biology) Deoxyuracil Nucleotides Poly A |
| Zdroj: | Journal of Virological Methods. 40:145-154 |
| ISSN: | 0166-0934 |
| DOI: | 10.1016/0166-0934(92)90063-j |
| Popis: | We developed a non-radioisotopic (non-RI) reverse transcriptase assay (RTA). The reverse transcriptase (RT) incorporates biotin-11-deoxyuridine-triphosphate (bio-dUTP) using a poly(rA) template hybridized with oligo(dT) primer that is immobilized on the surface of a 96-well microtiter plate. This assay is thus semi-automated by adapting it to an ELISA testing format. The incorporation of bio-dUTP was enhanced by adding cold dTTP to the reaction mixture, optimally in a molar ratio 4:1 (dTTP:bio-dUTP). This non-RI RTA is more sensitive than the conventional RI assay for the detection of purified Rous-associated virus 2 (RAV-2) and of human immunodeficiency virus type 1 (HIV-1) lysate. Because of its simple procedure, higher sensitivity and non-use of RI materials, the assay can be utilized not only for virological studies but also for routine safety screening of biological products for retroviral contamination. |
| Databáze: | OpenAIRE |
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