Yu H, Bruno J G, Cheng T C, Calomiris J J, Goode M T, Gatto-Menking D L
Optech Corporation, San Antonio, TX 78229, USA.
J Biolumin Chemilumin. 1995 Jul-Aug;10(4):239-45. doi: 10.1002/bio.1170100407.
Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)3(2+))-end-labelled primers. In this way, biotin for capture and Ru(bpy)3(2+) for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin- and Ru(bpy)3(2+)-labelled primers amplified a 1 kilobase region. Serial dilution of PCR product followed by magnetic separation with Streptavidin (SA)-coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection. ECL assay of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining. The combination of DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.
通过使用生物素化和钌（II）三联吡啶（Ru(bpy)3(2+)）末端标记引物的混合物，在聚合酶链反应（PCR）中可以实现靶DNA序列的扩增和检测。通过这种方式，用于捕获的生物素和用于检测的Ru(bpy)3(2+)直接掺入PCR产物中，从而避免了后续的探针杂交。使用生物素和Ru(bpy)3(2+)标记引物的混合物对交替单胞菌属菌株JD6.5的细菌DNA模板进行PCR扩增，得到了一个1千碱基的区域。对PCR产物进行系列稀释，然后用链霉亲和素（SA）包被的磁珠进行磁分离，并使用半自动QPCR系统5000进行电化学发光（ECL）检测，结果表明该方法对DNA的检测具有高灵敏度（皮克范围）。对与人类免疫缺陷病毒（HIV）序列的探针杂交进行ECL检测，也产生了皮克水平的灵敏度。通过ECL检测进行的DNA定量测定与电泳凝胶中DNA的肉眼检测结果具有良好的相关性。然而，通过ECL检测进行DNA检测的灵敏度比传统的溴化乙锭染色高10至100倍。基于DNA的磁分离与ECL检测相结合，提供了一种非常灵敏且快速的DNA定量方法，由于其快速简便的特性，可能在研究、环境监测、工业和临床领域有许多应用。
