Nakano T, Sano K, Odawara F, Saitoh Y, Otake T, Nakamura T, Hayashi K, Misaki H, Nakai M
Department of Microbiology, Osaka Medical College.
Kansenshogaku Zasshi. 1994 Jul;68(7):923-31. doi: 10.11150/kansenshogakuzasshi1970.68.923.
We developed an improved, highly sensitive non-radioisotopic (non-RI) reverse transcriptase (RT) assay (RTA). While the original non-RI method previously reported made use of primer immobilization, our improved method was based on a primer-template immobilization procedure. We tested the template specificity, reproducibility and linearity of the new method in assays of human immunodeficiency virus type-1 (HIV-1) RT. The sensitivities of the method previously reported, the improved method and the sensitive radioisotopic (RI-) RTA were compared in assays of recombinant HIV-1 RT, partially purified HIV-1 particles, and the culture supernatant derived from HIV-1-infected cells. For each of these samples except the culture supernatant the improved method was the most sensitive. It appeared that the fetal bovine serum presented in the culture medium interfered with the assay reaction. The curve describing inhibition of the assay reaction by fetal bovine serum showed that the highest degree of sensitivity in the assay was obtained when the culture supernatant sample was diluted four times. With this degree of dilution, the sensitivity of the new method for assay of culture supernatant sample was still half that of the sensitive RI-RTA. Culture supernatants of five peripheral blood mononuclear cell samples obtained from HIV-1-seropositive carriers were assayed by both the improved method and the sensitive RI-RTA; and with each of the methods, however all virus-positive cultures could be detected. The improved non-RI RTA was considered especially useful for assay of culture supernatants for purposes of virus isolation because of its advantages of excellent sensitivity and lack of requirement for radioisotopes.
我们开发了一种改进的、高灵敏度的非放射性同位素（non-RI）逆转录酶（RT）检测法（RTA）。虽然先前报道的原始非RI方法利用了引物固定化，但我们改进的方法基于引物-模板固定化程序。我们在人免疫缺陷病毒1型（HIV-1）RT检测中测试了新方法的模板特异性、重现性和线性。在重组HIV-1 RT、部分纯化的HIV-1颗粒以及HIV-1感染细胞来源的培养上清液检测中，比较了先前报道的方法、改进方法和灵敏的放射性同位素（RI-）RTA的灵敏度。对于除培养上清液外的这些样品中的每一种，改进方法都是最灵敏的。似乎培养基中存在的胎牛血清干扰了检测反应。描述胎牛血清对检测反应抑制作用的曲线表明，当培养上清液样品稀释4倍时，检测中获得了最高灵敏度。在此稀释度下，新方法检测培养上清液样品的灵敏度仍为灵敏的RI-RTA的一半。用改进方法和灵敏的RI-RTA对从HIV-1血清阳性携带者获得的5个外周血单个核细胞样品的培养上清液进行检测；然而，用每种方法都能检测到所有病毒阳性培养物。改进的非RI RTA因其优异的灵敏度和无需放射性同位素的优点，被认为对病毒分离目的的培养上清液检测特别有用。
