Zetterquist H, Olerup O
Center for BioTechnology, Karolinska Institute, NOVUM, Huddinge, Sweden.
Hum Immunol. 1992 May;34(1):64-74. doi: 10.1016/0198-8859(92)90086-3.
The clinical applicability of genomic HLA class II typing techniques has increased after the introduction of PCR-based typing strategies. In typing by PCR amplification using sequence-specific primers (PCR-SSP), amplification of specific alleles or groups of alleles is achieved, provided that the mismatch(es) of the SSP is located in the 3' end of the primer. Thus, the specificity of the typing system becomes part of the amplification step, which reduces the total typing time to a minimum by simplifying the postamplification processing of samples. The set of primers presented here identifies all of the alleles of the DR4 group, DRB10401-DRB10411, as well as the DRB107 and DRB10901 alleles. In the present study of DR4 alleles, PCR-SSP was compared with hybridization with sequence-specific oligonucleotide probes following group-specific PCR amplification (PCR-SSO). The two typing strategies gave completely concordant results in the 90 DR4-positive and the 32 DR4-negative individuals and cell lines studied. DR7,DQ9/DR9,DQ9 discrimination using PCR-SSP, was compared with MspI DQA RFLP typing, also with concordant results in the 33 DR7- and/or DR9-positive and 36 DR7- and DR9-negative individuals and cell lines tested. No false-negative or false-positive typing results were obtained. Genomic typing by PCR-SSP was performed in the overall time of 2 hours, including rapid DNA preparation, PCR amplification, postamplification processing, documentation, and interpretation of results. This makes the PCR-SSP strategy for HLA class II typing attractive not only in population- and disease-association studies, but also in routine clinical practice, including donor-recipient matching prior to cadaveric transplantation.
基于聚合酶链反应（PCR）的分型策略出现后，基因组HLA II类分型技术的临床适用性有所提高。在使用序列特异性引物进行PCR扩增分型（PCR-SSP）时，只要序列特异性引物的错配位于引物的3'端，就能实现特定等位基因或等位基因组的扩增。因此，分型系统的特异性成为扩增步骤的一部分，通过简化样本的扩增后处理，将总分型时间减至最短。此处展示的引物组可识别DR4组的所有等位基因，即DRB10401-DRB10411，以及DRB107和DRB10901等位基因。在本项关于DR4等位基因的研究中，将PCR-SSP与组特异性PCR扩增后与序列特异性寡核苷酸探针杂交（PCR-SSO）进行了比较。在研究的90例DR4阳性和32例DR4阴性个体及细胞系中，这两种分型策略得到的结果完全一致。使用PCR-SSP对DR7、DQ9/DR9、DQ9进行鉴别，并与MspI DQA限制性片段长度多态性（RFLP）分型进行比较，在检测的33例DR7和/或DR9阳性以及36例DR7和DR9阴性个体及细胞系中，结果也一致。未获得假阴性或假阳性分型结果。采用PCR-SSP进行基因组分型的总时长为2小时，包括快速DNA制备、PCR扩增、扩增后处理、记录及结果解读。这使得PCR-SSP策略用于HLA II类分型不仅在群体和疾病关联研究中具有吸引力，在常规临床实践中也很有吸引力，包括尸体移植前的供受者配型。
