Faas S J, Menon R, Braun E R, Rudert W A, Trucco M
Department of Pediatrics, University of Pittsburgh, School of Medicine, Pennsylvania, USA.
Tissue Antigens. 1996 Aug;48(2):97-112. doi: 10.1111/j.1399-0039.1996.tb02614.x.
Molecular typing of HLA DQB1 alleles, employing sequence-specific primers (SSP) for PCR amplification, was used to test a novel method that eliminates the requirement for subsequent gel electrophoresis or additional hybridization steps by directly detecting positive reactions. We have evaluated the performance of this fluorescence-based oligonucleotide probe assay to assign the most common DQB1 alleles on DNA from 14 homozygous cell lines and in a blind study of 50 diabetic patient samples that had been previously typed at the DQB1 locus using SSOP and conventional SSP-based approaches. We used a panel of 14 DQB1 SSP primer pairs, internal control primers, and a combination of 4 fluorescent oligonucleotide probes to detect 14 alleles or groups of alleles and controls. We can reliably detect single-base allelic differences, observe 100% concordance with the results obtained using both of the standard methods, and are able to further subtype several alleles that are not easily distinguished using SSOP (e.g. DQB1 *0401/0402 and DQB1 *0302/ 0303). Sequence-specific priming and exonuclease-released fluorescence (SSPERF) detection is technically simple and can be performed in less than 2 hours, including DNA extraction, PCR amplification, data analysis and allele identification. This method is particularly useful for the analysis of large numbers of samples, for which high throughput is critical and for which gel-based approaches are difficult to perform. This technique may also be useful for small-scale class I and class II molecular typing in clinically oriented laboratories.
采用序列特异性引物（SSP）进行PCR扩增，对HLA DQB1等位基因进行分子分型，以测试一种新方法，该方法通过直接检测阳性反应，无需后续的凝胶电泳或额外的杂交步骤。我们评估了这种基于荧光的寡核苷酸探针检测方法在14个纯合细胞系DNA上以及对50份糖尿病患者样本的盲测中的性能，这些样本先前已使用序列特异性寡核苷酸探针（SSOP）和传统的基于SSP的方法在DQB1位点进行过分型。我们使用了一组14对DQB1 SSP引物对、内部对照引物以及4种荧光寡核苷酸探针的组合来检测14个等位基因或等位基因组以及对照。我们能够可靠地检测单碱基等位基因差异，观察到与使用两种标准方法获得的结果100%一致，并且能够进一步对一些使用SSOP不易区分的等位基因进行亚型分析（例如DQB1 *0401/0402和DQB1 *0302/0303）。序列特异性引物延伸和核酸外切酶释放荧光（SSPERF）检测技术简单，包括DNA提取、PCR扩增、数据分析和等位基因鉴定在内，可在不到2小时内完成。该方法对于大量样本的分析特别有用，对于这些样本而言高通量至关重要，而基于凝胶的方法难以实施。该技术对于临床导向实验室中的小规模I类和II类分子分型也可能有用。
