Dahlén P O, Iitiä A J, Skagius G, Frostell A, Nunn M F, Kwiatkowski M
Pharmacia Genetic Engineering Inc., La Jolla, California 92037.
J Clin Microbiol. 1991 Apr;29(4):798-804. doi: 10.1128/jcm.29.4.798-804.1991.
The polymerase chain reaction (PCR) has many potential applications in the field of nucleic acid diagnostics. In particular, it has been successfully applied to the detection of pathogens present in low copy numbers such as the human immunodeficiency virus type 1. Here we describe a time-resolved fluorescence-based hybridization assay which, combined with the PCR, offers an extremely sensitive method for the detection of nucleic acids. In this assay format, the PCR is run by standard procedures and the subsequent hybridization reaction is carried out in solution by using two oligonucleotide probes, one biotinylated and one labeled with europium (Eu3+). The sandwich hybrids are then collected onto a streptavidin-coated microtitration well, and the bound Eu3+ is measured in a time-resolved fluorometer. This assay is rapid, user friendly, and quantitative and lends itself to automation. The application of this assay to the detection of human immunodeficiency virus type 1 is described.
聚合酶链反应（PCR）在核酸诊断领域有许多潜在应用。特别是，它已成功应用于检测低拷贝数存在的病原体，如1型人类免疫缺陷病毒。在此，我们描述一种基于时间分辨荧光的杂交检测方法，该方法与PCR相结合，为核酸检测提供了一种极其灵敏的方法。在这种检测形式中，PCR按标准程序进行，随后的杂交反应在溶液中通过使用两种寡核苷酸探针进行，一种用生物素标记，另一种用铕（Eu3+）标记。然后将夹心杂交体收集到包被链霉抗生物素蛋白的微量滴定孔上，并在时间分辨荧光计中测量结合的Eu3+。该检测方法快速、用户友好且定量，适合自动化操作。本文描述了该检测方法在1型人类免疫缺陷病毒检测中的应用。
