A non-radioisotopic reverse transcriptase assay using biotin-11-deoxyuridinetriphosphate on primer-immobilized microtiter plates.
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.
D001354:Avian Leukosis Virus / Q000201:enzymology / Q000302:isolation & purification; D009189:Avian Myeloblastosis Virus / Q000201:enzymology / Q000302:isolation & purification; D001710:Biotin / Q000031:analogs & derivatives*; D003856:Deoxyuracil Nucleotides; D015497:HIV-1 / Q000201:enzymology / Q000302:isolation & purification; D015498:HIV-2 / Q000201:enzymology / Q000302:isolation & purification; D009838:Oligodeoxyribonucleotides; D011061:Poly A; D012194:RNA-Directed DNA Polymerase / Q000032:analysis*; D012190:Retroviridae / Q000201:enzymology / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D014773:Virology / Q000379:methods
D003856:Deoxyuracil Nucleotides; D009838:Oligodeoxyribonucleotides; C027903:oligo (dT); D011061:Poly A; D001710:Biotin; C075017:5-(N-(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl)-2'-deoxuridine 5'-triphosphate; D012194:RNA-Directed DNA Polymerase