Sano K, Odawara F, Nakano T, Morimatsu S, Nakamura T, Saitoh Y, Jiang Y, Misaki H, Sakai Y, Nakai M
Department of Microbiology, Osaka Medical College, Japan.
J Virol Methods. 1995 Jun;53(2-3):235-44. doi: 10.1016/0166-0934(95)00028-s.
An improved non-radioisotopic (Non-RI) reverse transcriptase (RT) assay with a template-primer-immobilized microtiter plate is described, which has greater sensitivity than the former Non-RI RT assay previously described. Non-RI and commercially available non-radioactive (Non-RA) RT assays were compared for their ability to detect various polymerases. Two RTs from Rous-associated virus 2 (RAV-2) and avian myeloblastosis virus (AMV), one polymerase from Escherichia coli (Pol-I) and one recombinant RT of human immunodeficiency virus type 1 (HIV-1) were assessed. Two HIV-1 samples in a culture supernatant and pelleted virion suspended in Triton X-100 solution were measured. The Non-RI RT assay was one hundred times more sensitive by RAV-2 and Pol-I polymerases, and one thousand times more sensitive by the Non-RA assay than by the AMV RT. The Non-RI RT assay was 10, 16 and 64 times more sensitive than the Non-RA assay for measuring recombinant HIV-1 RT, pelleted virus and virus suspended in culture medium, respectively. To explain the discrepancy, it is shown that free biotin, such as in culture medium, disturbs the assay system of the Non-RA RT assay, but not the Non-RI assay. The present assay can be used to clarify the inhibitory mechanism of an anti-HIV-1 substance.
本文描述了一种改进的非放射性同位素（Non-RI）逆转录酶（RT）检测方法，该方法采用固定有模板引物的微量滴定板，比之前描述的非放射性同位素RT检测方法具有更高的灵敏度。比较了非放射性同位素和市售非放射性（Non-RA）RT检测方法检测各种聚合酶的能力。评估了来自劳斯相关病毒2（RAV-2）和禽成髓细胞瘤病毒（AMV）的两种逆转录酶、来自大肠杆菌的一种聚合酶（Pol-I）以及一种人类免疫缺陷病毒1型（HIV-1）重组逆转录酶。对培养上清液中的两份HIV-1样本以及悬浮于Triton X-100溶液中的病毒颗粒进行了检测。对于RAV-2和Pol-I聚合酶，非放射性同位素RT检测方法的灵敏度比非放射性检测方法高100倍，对于AMV逆转录酶，非放射性同位素RT检测方法的灵敏度比非放射性检测方法高1000倍。对于检测重组HIV-1逆转录酶、病毒颗粒以及悬浮于培养基中的病毒，非放射性同位素RT检测方法的灵敏度分别比非放射性检测方法高10倍、16倍和64倍。为了解释这种差异，研究表明，培养基中的游离生物素会干扰非放射性检测方法的检测系统，但不会干扰非放射性同位素检测方法。本检测方法可用于阐明抗HIV-1物质的抑制机制。
