HLA-DR TYPING BY PCR AMPLIFICATION WITH SEQUENCE-SPECIFIC PRIMERS (PCR-SSP) IN 2 HOURS - AN ALTERNATIVE TO SEROLOGICAL DR TYPING IN CLINICAL-PRACTICE INCLUDING DONOR-RECIPIENT MATCHING IN CADAVERIC TRANSPLANTATION
HLA-DR TYPING BY PCR AMPLIFICATION WITH SEQUENCE-SPECIFIC PRIMERS (PCR-SSP) IN 2 HOURS - AN ALTERNATIVE TO SEROLOGICAL DR TYPING IN CLINICAL-PRACTICE INCLUDING DONOR-RECIPIENT MATCHING IN CADAVERIC TRANSPLANTATION
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DOI：
10.1111/j.1399-0039.1992.tb01940.x
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发表时间：
1992-05-01
期刊：
影响因子：
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通讯作者：
ZETTERQUIST, H
中科院分区：
文献类型：
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作者：
OLERUP, O;ZETTERQUIST, H
在大多数基于聚合酶链式反应的组织分型技术中，聚合酶链式反应扩增之后是扩增后的特异性步骤。在用序列特异性引物进行聚合酶链式反应(PCR-SSP)分型时，分型的特异性是扩增步骤的一部分，这使得该技术几乎与血清组织分型一样快。在本研究中，设计了用于用PCR-SSP进行DR“低分辨率”分型的引物，即识别与血清学定义的系列DR1-DLu18相对应的多态性。这一决议是通过对每个个体进行19次PCR反应来实现的，其中17次用于指定DR1-DRw18，2次用于指定DRw52和DRwan53的超特异性。采用DR“低分辨率”PCR-SSP技术、TaqI DRB-DQA-DQB RFLP分析和血清学方法对30个细胞系和121个个体进行分型。PCR-SSP分型与RFLP分析的符合率为100%。在两次不同场合打印的40份样品中，重复性为100%。没有得到假阳性或假阴性的分型结果。所有DR1-DRV18纯合子和杂合子组合均可区分。扩增模式根据显性孟德尔遗传分离。DNA制备、PCR扩增和扩增后处理，包括凝胶检测、记录和解释，在2小时内完成。结论：PCR-SSP是一种灵敏度高、特异性强、重复性好的准确分型技术。该方法快速、廉价。用聚合酶链式反应-单链构象多态技术进行DR“低分辨率”分型非常适合于同时分析少量样本，在常规临床实践中是一种替代血清学DR分型的方法，包括身体移植中的供受者配型。
In most PCR-based tissue typing techniques the PCR amplification is followed by a post-amplification specificity step. In typing by PCR amplification with sequence-specific primers (PCR-SSP), typing specificity is part of the amplification step, which makes the technique almost as fast as serological tissue typing. In the present study primers were designed for DR "low-resolution" typing by PCR-SSP, i.e. identifying polymorphism corresponding to the serologically defined series DR1-DRw18. This resolution was achieved by performing 19 PCR reactions per individual, 17 for assigning DR1-DRw18 and 2 for the DRw52 and DRw53 superspecificities. Thirty cell lines and 121 individuals were typed by the DR "low-resolution" PCR-SSP technique, TaqI DRB-DQA-DQB RFLP analysis and serology. The concordance between PCR-SSP typing a nd RFLP analysis was 100%. The reproducibility was 100% in 40 samples typed on two separate occasions. No false-positive or false-negative typing results were obtained. All homozygous and heterozygous combinations of DR1-DRw18 could be distinguished. Amplification patterns segregated according to dominant Mendelian inheritance. DNA preparation, PCR amplification and post-amplification processing, including gel detection, documentation and interpretation, were performed in 2 hours. In conclusion, PCR-SSP is an accurate typing technique with high sensitivity, specificity and reproducibility. The method is rapid and inexpensive. DR "low-resolution" typing by the PCR-SSP technique is ideally suited for analyzing small numbers of samples simultaneously and is an alternative to serological DR typing in routine clinical practice including donor-recipient matching in cadaveric transplantations.