套式序列特异引物聚合酶链反应技术的建立及其在HLA配型中的应用

Development of a nested PCR-SSP method and its application in HLA typing

建立套式序列特异引物聚合酶链反应（ＰＣＲ－ＳＳＰ）方法，用于ＨＬＡ－ＤＲ基因分型。利用一对外引物扩增ＨＬＡ－ＤＲＢ１第２外显子部分片段（２８３ｂｐ），作为第２次扩增的模板，ＰＣＲ循环参数：９８℃、５分钟；９４℃、６０秒，７２℃、３０秒，６３℃、３０秒，１５个循环；利用各组特异性引物进行第２次扩增，产生与血清学相对应的ＨＬＡ－ＤＲ型别，ＰＣＲ循环参数：９４℃、３０秒，７２℃、３０秒，６０℃、３０秒，２０个循环。结果表明本方法特异性好。在７个家庭２０例受试者中进行了应用，与血清学方法比较，两方法确定出的型别，８０％一致，２０％不同，另有血清学为“空”的８个型别，基因分型全部为ＤＲ９阳性。

A nested PCR sequence specific primer (PCR-SSP) method was developed to be used in HLA-DR genotyping. A pair of primers was used to amplify a 283bp fragment of HLADRB1 exon 2, then 11 pairs of groupspecific primers were utilized to amplify the corresponding fragments by which HLA-DR specificities were determined. A total of 20 DNA samples from patients were analyzed for HLA matching before bone marrow transplantation by both serological and genetic methods. The results indicated that the same types were identified in 80%, different types in 20% by the two methods. In the 20 DNA samples, 7 kinds of types: DR2, DR36, DR4, DR7, DR9, DR11, DR12 were amplified.