HLA-DQB1 AND HLA-DQA1 TYPING BY PCR AMPLIFICATION WITH SEQUENCE-SPECIFIC PRIMERS (PCR-SSP) IN 2 HOURS
HLA-DQB1 AND HLA-DQA1 TYPING BY PCR AMPLIFICATION WITH SEQUENCE-SPECIFIC PRIMERS (PCR-SSP) IN 2 HOURS
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DOI：
10.1111/j.1399-0039.1993.tb01991.x
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发表时间：
1993-03-01
期刊：
影响因子：
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通讯作者：
FOGDELL, A
中科院分区：
文献类型：
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作者：
OLERUP, O;ALDENER, A;FOGDELL, A
在本研究中，设计了用于检测所有表型表达的DQB1和DQA1等位基因变异性的聚合酶链式反应引物，分别为19个和10个等位基因。对于DQB1分型，每个样本由第一组14对PCR引物扩增，在某些情况下，随后进行2至6次额外的PCR反应。前14对引物允许鉴定/分离除少数最近描述的DQB1等位基因以外的所有等位基因：DQB1*0504、DQB1*0605、DQB1*0606和DQB1*0607将无法识别；DQB1*0603和DQB1*0608；以及DQB1*0301和DQB1*0304将分别无法区分。因此，另外一组8对DQB1引物被用来进行完整的DQB1分型，包括所有纯合和杂合组合。对于DQA1分型，每个样本进行了12次聚合酶链式反应，其中10次用于检测第二外显子内的变异，两次用于鉴定第一外显子的多态性。所有纯合和杂合的DQA1等位基因组合都能被这些引物分辨出来。此外，还设计了4个用于测定人类白细胞抗原-DQB1基因57密码子的引物。用DQB1、DQA1、DQA1、DQB_(57)引物对30个细胞系和120个个体进行了检测。PCR-SSP分型与TaqI DRB-DQA-DQB RFLP分析的DQB1、DQA1等位基因分型的符合率为100%。在两次不同场合调查的30个样品中，重复性为100%。研究了15个核心家系的扩增模式，根据显性孟德尔遗传进行分离。DQB1和DQA1PCR-SSP分型可在2小时内完成，包括DNA提取、PCR扩增和扩增后处理。这种方法在技术上很简单，类型也很容易解释。对一个个体进行分型的成本很低，而且与同时分析的样本数量无关，即该技术非常适合常规临床使用。
In the present study PCR primers were designed for detecting all phenotypically expressed DQB1 and DQA1 allelic variability, 19 and 10 alleles, respectively, by PCR amplification with sequence-specific primers (PCR-SSP). For DQB1 typing, each sample was amplified by a first set of 14 PCR primer pairs, followed in some cases by two to six additional PCR reactions. The first 14 primer pairs allowed the identification/separation of all but a few of the recently described DQB1 alleles: DQB1*0504, DQB1*0605, DQB1*0606 and DQB1*0607 would not be identified; DQB1*0603 and DQB1*0608; and DQB1*0301 and DQB1*0304, respectively, would not be distinguished. Therefore an additional set of eight DQB1 primer pairs was used for a complete DQB1 typing, including all homozygous and heterozygous combinations. For DQA1 typing, 12 PCR reactions were performed per sample, 10 for detecting variability within the second exon and two for identifying first exon polymorphism. All homozygous and heterozygous combinations of DQA1 alleles could be resolved by these primer pairs. In addition, four primer mixes were designed for determining codon 57 of the HLA-DQB1 gene. Thirty cell lines and 120 individuals were investigated by the DQB1 and DQA1 PCR-SSP technique, as well as with the HLA-DQbeta57 primers. The concordance between PCR-SSP typing and assigning DQB1 and DQA1 alleles from TaqI DRB-DQA-DQB RFLP analysis was 100%. The reproducibility was 100% in 30 samples investigated on two separate occasions. Amplification patterns, investigated in 15 nuclear families, segregated according to dominant Mendelian inheritance. DQB1 and DQA1 PCR-SSP typing can be performed in 2 hours, including DNA extraction, PCR amplification and post-amplification processing. The method is technically simple and the typings are easy to interpret. The cost for typing one individual is low and is independent of the number of samples analyzed simultaneously, i.e. the technique is well-suited for routine clinical use.