Suzuki K, Craddock B P, Okamoto N, Kano T, Steigbigel R T
Biomedical Research Center, Olympus Corporation, East Setauket, NY 11733.
Biotechnol Appl Biochem. 1993 Aug;18(1):37-44.
A colorimetric assay for detection of reverse transcriptase (RT) of the human immunodeficiency virus (HIV) was developed using oligodeoxythymidylic acid (oligo-dT)-linked magnetic beads and digoxigenin-deoxyuridine triphosphate (dig-dUTP). During the RT reaction, dig-dUTP was incorporated into oligo-dT which had been hybridized to polyadenylic acid [poly (A)]. At the detection step, an alkaline phosphatase-conjugated antibody to digoxigenin was added, followed by the addition of a colorimetric substrate for this enzyme. This method showed excellent correlation with the isotopic RT assay, which used tritiated thymidine triphosphate ([3H]dTTP), for detection of purified avian-myeloblastosis-virus RT (AMV-RT). This assay also demonstrated close correlation with the isotopic RT assay using human peripheral-blood lymphocytes infected in vitro with HIV. This colorimetric RT assay offers important advantages over the conventional radioactive RT assays with respect to its simplicity, safety and cost. The total assay time, including the RT reaction step, was less than 1 h, and therefore provides a reliable rapid assay for detection and quantification of HIV.
利用与寡聚脱氧胸苷酸（oligo-dT）连接的磁珠和地高辛配基-脱氧尿苷三磷酸（dig-dUTP），开发了一种用于检测人类免疫缺陷病毒（HIV）逆转录酶（RT）的比色测定法。在逆转录反应过程中，dig-dUTP掺入到已与聚腺苷酸[poly（A）]杂交的oligo-dT中。在检测步骤中，加入与地高辛配基结合的碱性磷酸酶抗体，随后加入该酶的比色底物。该方法与使用氚标记的胸腺嘧啶三磷酸（[3H]dTTP）检测纯化的禽成髓细胞瘤病毒逆转录酶（AMV-RT）的同位素逆转录测定法具有良好的相关性。该测定法还证明与使用体外感染HIV的人外周血淋巴细胞的同位素逆转录测定法密切相关。这种比色逆转录测定法在简便性、安全性和成本方面比传统的放射性逆转录测定法具有重要优势。包括逆转录反应步骤在内的总测定时间不到1小时，因此为HIV的检测和定量提供了一种可靠的快速测定法。
