Genotyping of platelet antigens HPA-2 with melting curve analysis

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 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 Results 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 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). 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