一种在引物固定的微量滴定板上使用生物素-11-脱氧尿苷三磷酸的非放射性逆转录酶测定法。

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

作者信息

Urabe T, Sano K, Tanno M, Mizoguchi J, Otani M, Lee M H, Takasaki T, Kusakabe H, Imagawa D T, Nakai M

机构信息

Department of Microbiology, Osaka Medical College, Japan.

出版信息

J Virol Methods. 1992 Nov;40(2):145-54. doi: 10.1016/0166-0934(92)90063-j.

DOI:10.1016/0166-0934(92)90063-j

PMID:1280640

Abstract

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.

摘要

我们开发了一种非放射性同位素（non-RI）逆转录酶检测法（RTA）。逆转录酶（RT）利用与固定在96孔微量滴定板表面的寡聚（dT）引物杂交的聚（rA）模板掺入生物素-11-脱氧尿苷三磷酸（bio-dUTP）。通过将其调整为ELISA检测形式，该检测法实现了半自动化。通过向反应混合物中添加冷dTTP，最佳摩尔比为4:1（dTTP:bio-dUTP），可增强bio-dUTP的掺入。这种非RI RTA在检测纯化的劳斯相关病毒2（RAV-2）和人类免疫缺陷病毒1型（HIV-1）裂解物方面比传统的RI检测法更灵敏。由于其操作简单、灵敏度高且不使用RI材料，该检测法不仅可用于病毒学研究，还可用于生物制品逆转录病毒污染的常规安全筛查。