熔解曲线分析法用于RHD 1227G>A基因分型的实验研究  

Genotyping of RHD gene 1227 G>A by melting curve analysis

作　　者：王霓[1] 周世航[1] 邵林楠 于卫建[1] 张开立[2] 刘铭[2] WANG Ni;ZHOU Shihang;SHAO Linnan;YU Wei-jian;ZHANG Kaili;LIU Ming(Department of Blood Group,Dalian Blood Center,116001;Department of Cellular Biol-ogy,Dalian Medical University.)

机构地区：[1]大连市血液中心,辽宁大连116001 [2]大连医科大学细胞生物学教研室

出　　处：《中国输血杂志》2018年第9期953-956,共4页Chinese Journal of Blood Transfusion

摘　　要：目的建立用于RHD 1227G〉A基因分型的熔解曲线分析法。方法设计合成RHD 1227G〉A等位基因特异性引物和公共引物,并在2条等位基因特异性引物5＇端加上不同长度的GC序列。在实时PCR反应后,进行熔解曲线分析,依据PCR产物熔解温度的差异,对RHD 1227G〉A基因分型。将建立的熔解曲线分析法与常规的PCRSSP法比较。对于2种方法检测结果不符的标本,进行RHD基因第9外显子测序,确定该标本为RHD 1227G〉A基因型。结果应用熔解曲线分析法对84例标本进行基因分型,其中RHD 1227A＋/G-32例,1227A-/G＋22例,1227A＋/G＋6例,1227A-/G-24例。发现2例标本熔解曲线法检测的基因型为1227A＋/G-,而PCR-SSP法检测结果为1227A＋/G＋。经基因测序证实这2例标本结果是1227A＋/G-。结论用于RHD 1227G〉A基因分型的熔解曲线法具有操作简单、快速、准确等优点,优于常规的PCR-SSP法。Objective To develop a method for RHD 1227G 〉A genotyping using melting curve analysis （MCA）. Methods Two allele-specific primer for RHD 1227G〉A and a common primer were designed and synthesized. Two GC-rich tails of different lengths were attached to 5＂ends of the 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,RHD 1227G〉A could be genotyped. All genotyping results were compared with the results obtained with con- ventional PCR-SSP. For the discordant results, RhD exon 9 sequencing was performed to determine the final RHD 1227G〉A genotype. Results A total of 84 samples were genotyped for RHD 1227G〉A by MCA. 32 samples were typed as 1227A＋/G -, 22 samples were typed as 1227A-/G＋, 6 samples were typed as 1227A＋/G＋ and 24 samples were typed as 1227A-/G -. Two samples were typed as 1227A＋/G＋ by PCR-SSP but 1227A＋/G- by MCA, which were further confirmed to be1227A＋/G- by RHD by exon 9 sequencing. Conclusion MCA for RHD 1227G〉A genotyping is simple, rapid, accurate, and it is superior to conventional PCR-SSP.

关 键 词：实时PCR 熔解曲线分析 D^(el) RHD基因 RHD 1227A等位基因 基因分型 

分 类 号：R457.11[医药卫生—治疗学]