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为了探讨通过变性高效液相色谱(DHPLC)进行HLA-DRB1配型的可行性,选择经PCR-SSP证实的20例HLA-DRB1相合及2例HLA-DRB1不相合的病人及其同胞供者的标本为实验对象,全部标本通过PCR扩增HLA-DRB1第二外显子基因片段,PCR产物经梯度变性处理后通过DHPLC检测,在部分变性温度下,检测病人与供者的HLA-DRB1第二外显子DHPLC峰型是否相同,以判断供受者的HLA-DRB1基因型是否相同。结果表明DHPLC与PCR-SSP进行DRB1配型比较分析,经kappa检验,kappa值为0.776,P值=0.00,说明两种方法的配型结果无显著性差别。结论变性高效液相色谱方法用于HLA-DRB1配型方便,经济实用并解决了HLA新基因的不断出现与相应的位点的引物或探针设计和开发的相对滞后之间的矛盾。
To investigate the feasibility of HLA-DRB1 typing by denaturing high performance liquid chromatography (DHPLC), 20 HLA-DRB1-matched and 2 HLA-DRB1-incompatible patients and their siblings confirmed by PCR-SSP were selected The specimens were used as the experimental subjects. All the specimens amplified the second exon fragment of HLA-DRB1 by PCR. The PCR products were detected by DHPLC after gradient denaturation. Second, the HLA-DRB1 Exon DHPLC peak patterns are the same, in order to determine the donor’s HLA-DRB1 genotypes are the same. The results showed that DHPLC and PCR-SSP comparison of DRB1 typing, kappa test, kappa value of 0.776, P = 0.00, indicating no significant difference between the two methods of matching results. Conclusion The method of denaturing high performance liquid chromatography for HLA-DRB1 matching is convenient, economical and practical and solves the contradiction between the appearance of new HLA genes and the relative lag of the primer or probe design and development at the corresponding sites.