血小板抗原HPA-2基因分型熔解曲线法分析

Genotyping of platelet antigens HPA-2 with melting curve analysis

目的建立熔解曲线分析法进行HPA-2基因分型,并检测大连地区汉族人群HPA-2等位基因频率。方法实时荧光PCR后用熔解曲线分析法进行HPA-2基因分型,与常规PCR-SSP方法进行比较,用熔解曲线分析法对辽宁地区155名汉族血小板捐献者的HPA-2系统进行基因分型,获得HPA-2等位基因分布数据,并与国内部分地区的人群进行比较。结果基因型为HPA-2aa的样本,熔解曲线呈现单一的峰型,Tm值在(85±1)℃;基因型为HPA-2ab的样本熔解曲线呈现双峰型,Tm值在(80±1)℃和(85±1)℃;基因型为HPA-2bb的样本熔解曲线呈现单一的峰型,Tm值在(80±1)℃。熔解曲线分析法基因分型结果与PCR-SSP法完全一致。大连地区汉族人群HPA-2a、HPA-2b等位基因频率分别为0.9161,0.0838。与南京、上海、新疆地区汉族人群以及新疆地区维吾尔族人群均很相似(P>0.05),但与海南黎族人群相比差异有统计学意义(P<0.05)。结论应用熔解曲线分析法进行HPA-2基因分型具有简单、快速、准确等优点,优于常规的PCR-SSP方法;HPA-2等位基因频率分布在国内存在种族差异。

Objective To develop a genotyping method for human platelet antigens-2 （HPA-2）by using melting curve analysis and to investigate the gene frequencies of HPA-2 in Han population in Dalian city. Methods Real-time PCR was used and melting curve analysis was performed for genotyping of HPA-2. Reproducibility of the method was evaluated and the results of melting curve analysis were compared with those using traditional PCR amplification with se- quence-specific primers（ PCR-SSP） method. The genotype of HPA-2 in 155 Hart platelet donors were determined using melting curve analysis,then the gene frequencies of HPA-2 were compared with that of other populations in China Re- suits Melting curve of HPA-2aa sample showed a single peak with the melting temperature（Tm） of 85 ±1℃ ;melting curve of HPA-2ab sample showed two peaks with the Tm of 80 ±1 ℃ and 85 ±1 ℃ ; and melting curve of HPA-2bb sample showed a single peak, with the Tm of 80 ±1 ℃. There was complete concordance of results for all samples tested by PCR-SSP and melting curve analysis. The gene frequencies of HPA-2a and HPA-2b in Hart populations of Dalian city were 0. 9161 and 0. 0838 ,respectively. The distribution of HPA-2 alleles in Liaoning Han populations is simi- lar to that in Nanjing, Shanghai, and Xinjiang Han and Xingjiang Uyghur populations （P 〉 0.05 for all）, but it significant- ly differ from that in Hainan Li national minority people （ P 〈 0. 05 ）. Conclusion The method for genotyping of HPA-2 by using melting curve analysis was developed and the method is faster, simpler, and more accurate compared to the results of traditional PCR-SSP. There are differences in HPA-2 genotypes among different race in China