Sjöroos M, Ilonen J, Reijonen H, Lövgren T
Department of Biotechnology, University of Turku, Finland.
Dis Markers. 1998 Jun;14(1):9-19. doi: 10.1155/1998/350145.
A microtitration plate based time-resolved fluorescence (TRF) hybridisation assay was developed for HLA typing utilising biotinylated sequence-specific catching probes and europium (Eu) labelled gene locus-specific detection probe to allow time-resolved fluorometer reading of the reaction. In an application for HLA-DQA typing a 228 base pair long region of the polymorphic exon 2 of DQA1 gene was amplified and the denatured PCR product distributed into streptavidin-coated microtitration wells together with the detection probe and one of the catching probes. After incubation and washes, the enhancement solution was added and specific hybridisation signal detected by measuring the emitted light. A series of 100 isolated genomic DNA samples were studied using biotinylated probes specific for DQA1*01, *0101/0104, *0103/0201/0601, *0201, *03, *0401/0601, *05 and *0502 alleles with results demonstrating the capacity of the test to detect aimed alleles. A series of whole blood spot samples were also studied and the results confirmed the applicability of this modification of the test.
开发了一种基于微量滴定板的时间分辨荧光（TRF）杂交检测法用于HLA分型，该方法利用生物素化的序列特异性捕获探针和铕（Eu）标记的基因座特异性检测探针，以便用时间分辨荧光计读取反应。在HLA - DQA分型的应用中，扩增了DQA1基因多态性外显子2的一个228个碱基对长的区域，并将变性的PCR产物与检测探针和一种捕获探针一起分配到链霉亲和素包被的微量滴定孔中。温育和洗涤后，加入增强溶液，并通过测量发射光检测特异性杂交信号。使用针对DQA1*01、*0101/0104、*0103/0201/0601、*0201、*03、*0401/0601、05和0502等位基因的生物素化探针研究了一系列100个分离的基因组DNA样本，结果证明了该检测方法检测目标等位基因的能力。还研究了一系列全血斑点样本，结果证实了该检测方法这种改进的适用性。
