熔解曲线分析法用于HPA-15基因分型的研究

目的探讨熔解曲线分析法用于HPA-15基因分型,了解大连地区汉族人群HPA-15基因多态性。方法设计合成HPA-15等位基因特异性引物和1条公共引物,其中在2条等位基因特异性引物的5'末端分别加入具有不同长度并富含GC碱基的序列。使用这3条引物,进行实时PCR反应。在PCR反应后,进行熔解曲线分析,依据PCR产物熔解温度的差异,进行基因分型。应用熔解曲线分析法和常规PCR-SSP法对100名大连地区汉族人群的血液标本进行HPA-15基因分型。此外对熔解曲线分析法的重复性进行评价。结果 100个血液标本的熔解曲线分析法和常规PCR-SSP法的基因分型结果是完全一致的。10个血标本的2次熔解曲线分析结果是一致的。大连地区汉族人群HPA-15a、-15b基因频率分别为0.58和0.42。大连地区汉族人群与国内部分地区汉族人群相比,HPA-15等位基因频率的差异无统计学意义(P>0.05)。与新疆维吾尔族人群相比,HPA-15等位基因频率差异有统计学意义(P<0.05)。结论 HPA的熔解曲线基因分型方法具有快速、简单、准确、通量高、重复性好等优点,要优于常规的PCR-SSP法。在国内HPA-15等位基因分布存在着种族差异。

Genotyping of human platelet antigen 15 by melting curve analysis

Objective To investigate genotyping of human platelet antigen 15(HPA-15)by using melting curve analysis,and study the polymorphism of human platelet antigen 15 in Dalian Han population.Methods Two HPA-15 allele-specific primer and a common primer were designed and synthesized.Two GC-rich tails of different lengths were attached to 5’end of HPA-15 allele-specific PCR primers.Real-time PCR was carried out with the three primers.After real-time PCR,melting curve analysis was performed.By differences of the melting temperatures of the PCR products,HPA-15 can be genotyped.One hundred blood samples were genotyped for HPA-15 by the melting curve analysis and conventional PCR-SSP.Moreover,the reproducibility of melting curve analysis was also assessed.Results There was complete concordance of results for 100 samples tested by melting curve analysis and PCR-SSP.The retesting results of melting curve analysis were consistent with those of the initial testing for 10 samples.Gene frequencies of HPA-15a and HPA-15b in Dalian Han population were 0.580 0 and 0.420 0 respectively.The distributions of HPA-15 in Dalian Han population did not differ significantly from that in some China Han population(P>0.05),but did differ significantly from that in China Uighurs population(P<0.05).Conclusion The melting curve analysis for HPA-15 genotyping is rapid,simple,accurate,high-throughput and reproducible and it is superior to conventional PCR-SSP.There were different gene frequencies for HPA-15 alleles among different races of China.