A NONRADIOISOTOPIC REVERSE-TRANSCRIPTASE ASSAY USING BIOTIN-11-DEOXYURIDINETRIPHOSPHATE ON PRIMER-IMMOBILIZED MICROTITER PLATES

作者：URABE T; SANO K; TANNO M; MIZOGUCHI J; OTANI M; LEE MH; TAKASAKI T; KUSAKABE H; IMAGAWA DT; NAKAI M

来源：Journal of Virological Methods, 1992, 40(2): 145-154.

DOI：10.1016/0166-0934(92)90063-J

• BIOTIN-11-DEOXYURIDINETRIPHOSPHATE
• ROUS-ASSOCIATED VIRUS-2
• HUMAN IMMUNODEFICIENCY VIRUS
• REVERSE TRANSCRIPTASE ASSAY

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.

• 出版日期1992-11