Genotyping of platelet antigens HPA-2 with melting curve analysis

ObjectiveTo 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.MethodsReal-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 sequence-specific primers(PCR-SSP)method.The genotype of HPA-2 in 155 Han platelet donors were determined using melting curve analysis,then the gene frequencies of HPA-2 were compared with that of other populations in China ResultsMelting 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 Han populations of Dalian city were 0.9161 and 0.0838,respectively.The distribution of HPA-2 alleles in Liaoning Han populations is similar to that in Nanjing,Shanghai,and Xinjiang Han and Xingjiang Uyghur populations(P>0.05 for all),but it significantly differ from that in Hainan Li national minority people(P<0.05).ConclusionThe 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.