A non-radioisotopic reverse transcriptase assay using biotin-11-deoxyuridinetriphosphate on primer-immobilized microtiter plates

• Published in: Journal of virological methods Vol. 40; no. 2; pp. 145 - 154
• Main Authors: Urabe, Takeshi, Sano, Kouichi, Tanno, Masashi, Mizoguchi, Junzo, Otani, Masaru, Lee, Moon H., Takasaki, Tomohiko, Kusakabe, Hidenari, Imagawa, David T., Nakai, Masuyo
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 01.11.1992; Amsterdam Elsevier; New York, NY
• Subjects: AIDS/HIV; Avian Leukosis Virus - enzymology; Avian Leukosis Virus - isolation & purification; Avian Myeloblastosis Virus - enzymology; Avian Myeloblastosis Virus - isolation & purification; Biological and medical sciences; Biotin - analogs & derivatives; Biotin-11-deoxyuridinetriphosphate; Deoxyuracil Nucleotides; Fundamental and applied biological sciences. Psychology; HIV-1 - enzymology; HIV-1 - isolation & purification; HIV-2 - enzymology; HIV-2 - isolation & purification; Human immunodeficiency virus; Microbiology; Oligodeoxyribonucleotides; Poly A; Retroviridae - enzymology; Retroviridae - isolation & purification; Reverse transcriptase assay; RNA-Directed DNA Polymerase - analysis; Rous-associated virus 2; Sensitivity and Specificity; Templates, Genetic; Virology; Virology - methods; Rous-associated virus 2; Reverse transcriptase assay; Human immunodeficiency virus; Biotin-11-deoxyuridinetriphosphate; Virus; Enzyme; DNA polymerase RNA-dependent; Retroviridae; Lentivirinae; Detection
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/0166-0934(92)90063-J

We developed a non-radioisotopic (non-RI) reverse transcriptase assay (RTA). The reverse transcriptase (RT) incorporates biotin-11-deoxyuridinetriphosphate (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.