PCR－SSP快速HLA－DR2、DR7、DR9基因配型研究  

Rapid genotyping for HLA-DR2, DR7,DR9 by PCR amplification with sequence-specific primers

作　　者：谭建明[1] 谢桐[1] 徐琴君[1] 王祥慧[1] 丁言德[1] 

机构地区：[1]上海市第一人民医院泌尿外科研究室

出　　处：《中华泌尿外科杂志》1995年第5期262-264,共3页Chinese Journal of Urology

基　　金：国家自然科学基金

摘　　要：采用顺序特异性引物聚合酶链式反应（ＰＣＲ－ＳＳＰ）ＤＮＡ分型技术，首次对３５例肾移植供受者和４份标准ＤＮＡ进行ＨＬＡ－ＤＲ２、ＤＲ７、ＤＲ９基因配型。ＰＣＲ扩增条件为９４℃变性３０秒，６０℃退火５０秒，７２℃延伸４０秒，５个循环后降低退火温度至５８℃共３４个循环，即可在凝胶电泳上出现清晰可辨的特异性产物和阳性对照条带。经分子量标志初步鉴定，标准ＤＮＡ和地高辛标记的特异性探针Ｓｏｕｔｈｅｒｎ杂交确定性鉴定证实。实验结果稳定可靠，特异性和重复性达１００％，无假阳性和假阴性，耗时５小时。显示ＰＣＲ－ＳＳＰ基因配型具有高分辨度、高特异性和简捷快速的特点，适合于临床应用．HLA-DR2, DR7, DR9 genotyping by polymerase chain reaction with sequence-specific primers (PCR-SSP)was carried out for 35 individuals and 4 cell lines DNA. The PCR cycling conditions were as follows : 5 cycles denaturation at 94℃ for 30s, annealing at 60℃ for 50s and extension at 72 ℃ for 40s followed immediatedly by 29 cycles at 94℃for 30s,at 58℃ for 50s and at 72℃for 40s. The specific PCR products and internal positive control were clearly visualized on agarose gel electrophoresis. The specificity of the products were determined against molecular marker, a panel of standard DNA representing DR15,DR16,DR7,DR9 and Southern hybridization using DIG oligonucleotide 3'end labeling probes.The reproducibility was 100%,false-positive or false-negative typing results were observed. Genotyping by PCR-SSP could be accomplished in the overall time of 5 hours. The results showed that genotyping for HLA-DR by PCR-SSP has proved to be a high-sensitivity, high-specificity and rapid technique,suited for clinical practice including donor-recipient matching prior to cadaveric transplantation.

关 键 词：肾移植 聚合酶链式反应 人类白细胞抗原 

分 类 号：R699.2[医药卫生—泌尿科学]