多重聚合酶链反应-微流芯片检测人血小板抗原系统基因型方法的建立与应用

The foundation and application a genotyping method of human platelet antigen systems with multiplex PCS and microfluidic chip assay

目的建立人血小板抗原(HPA)-2、4、5系统基因型的检测方法。方法用多重聚合酶链反应和微流芯片检测方法,通过设计9条特异性引物、优化反应体系和反应条件,在一个体系中同时扩增HPA-2、4、5系统特异性的目的基因片段,利用微流芯片快速检测HPA-2、4、5系统基因型;并把分型结果与采用聚合酶链反应-序列特异性引物(PCR-SSP)获得的结果进行比较。结果对35名健康单采血小板者进行HPA-2、4、5系统分型的结果为:33名为2a／2a型,2名为28／2b型;34名为4a／4a型,1名为4a／4b型;29名为5a／5a型,6名为,5a／5b型。未发现2b／2b、4b／4b及5b／5b纯合子个体。该结果与PCR-SSP方法获得的结果完全一致。结论该方法可快速、准确地用于血小板血型抗原基因分型,尤其适合于大样本检测。

Objective To establish the method for simultaneous genotyping of the human platelet antigen （HPA）-2,4,5 systems. Methods With the multiplex PCR and the microfluidic chip assay, nine specific primers were designed and the reaction system and condition were optimized for the simultaneous amplification of the specific target gene segments of the HPA-2, 4, 5 systems in one system and the microfluidic chip assay was used for the rapid genotyping of the HPA-2, 4, 5 systems. The results of genotyping were compared with those obtained with the polymerase chain reaction method using sequence specific primers （PCR-SSP）. Results The results of HPA 2,4,5 systems in 35 healthy platelet donors were as follows：2a/2a in 33 donors and 2a/2b in 2; 4a/4a in 34 and 4a/4b in 1 ; 5a/5a in 29 and 5a/5b in 6. The 2b/2b, 4b/4b and 5b/5b homozygous individuals were found in no donors. The results were completely in accord with those obtained with PCR-SSP. Condusion The method of multiplex PCR and microfluidic chip assay can be used for the rapid and accurate genotyping of HPA, especially for the determination in large samples.