Donovan R M, Bush C E, Peterson W R, Parker L H, Cohen S H, Jordan G W, Vanden Brink K M, Goldstein E
Department of Internal Medicine, University of California, Davis 95616.
Mol Cell Probes. 1987 Dec;1(4):359-66. doi: 10.1016/0890-8508(87)90017-x.
Simple and sensitive methods to directly detect the human immunodeficiency virus (HIV) are needed for routine use in the clinical laboratory. In this study, we compared DNA probes prepared by: (1) nick translation with biotinylated dATP; (2) direct covalent biotinylation with photobiotin; (3) direct covalent reaction with 2-acetylaminofluorene (AAF); and (4) a standard radioactive (32P) nick translation procedure. These four DNA probes were hybridized with dilutions of purified target HIV DNA blotted onto nitrocellulose strips. Hybridization was detected using a complex of strepavidin-alkaline phosphatase [for (1) and (2)], alkaline phosphatase-tagged antibodies [for (3)] and by autoradiography [for (4)]. Alkaline phosphatase was detected colorimetrically using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. After 1 h, AAF probes were most sensitive (amount detected less than 5 pg), followed by biotin (10 pg), photobiotinylated probes (20 pg) and the radioactive probe (10 pg). The AAF probes were then used to detect HIV DNA in infected CEM cells. We conclude that non-radioactive DNA labelling methods can be used to directly detect HIV DNA under conditions compatible with present clinical laboratory procedures.
临床实验室的常规检测需要简单、灵敏的直接检测人类免疫缺陷病毒（HIV）的方法。在本研究中，我们比较了通过以下方法制备的DNA探针：（1）用生物素化的dATP进行缺口平移；（2）用光生物素进行直接共价生物素化；（3）与2-乙酰氨基芴（AAF）进行直接共价反应；（4）标准放射性（32P）缺口平移程序。将这四种DNA探针与点样在硝酸纤维素膜条上的纯化目标HIV DNA稀释液进行杂交。使用链霉亲和素-碱性磷酸酶复合物[用于（1）和（2）]、碱性磷酸酶标记的抗体[用于（3）]以及放射自显影[用于（4）]检测杂交情况。使用硝基蓝四唑和5-溴-4-氯-3-吲哚磷酸通过比色法检测碱性磷酸酶。1小时后，AAF探针最灵敏（检测量小于5 pg），其次是生物素（10 pg）、光生物素化探针（20 pg）和放射性探针（10 pg）。然后使用AAF探针检测感染的CEM细胞中的HIV DNA。我们得出结论，在与当前临床实验室程序兼容的条件下，非放射性DNA标记方法可用于直接检测HIV DNA。
