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目的应用高分辨熔解曲线分析技术检测中国人常见β地中海贫血基因突变。方法 PCR扩增HBB基因3个外显子、外显子-内含子交界区及第2内含子部分区域,采用LightScanner对PCR产物进行高分辨熔解曲线分析,通过DNA;;测序对HRM结果进行验证。结果对48例正常对照的HRM分析显示,在HBB基因的突变筛查区域存在两个常见SNPs;在验证实验中,对37例已知基因型患者的HRM分析显示,所有突变均得到正确检测且每种基因型都有其独特的熔解曲线;在双盲实验中,对70例未知样本的HRM分析共检出28个异常熔解曲线,与测序结果完全一致,HRM检测的灵敏度和特异性均为100%。结论高分辨熔解曲线分析法检测HBB基因突变具有简单快速、高效敏感、成本低廉等优点,不仅可用于β地贫的突变筛查,还可对已知突变进行基因分型。
Objective To apply high resolution melting curve analysis to detect common β thalassemia mutations in Chinese. Methods Three exons of HBB gene, exon-intron junction and part of intron 2 of HBB gene were amplified by PCR. The PCR products were analyzed by high resolution melting curve using LightScanner. authenticating. Results HRM analysis of 48 normal controls revealed two common SNPs in the mutation screening area of the HBB gene. In validation experiments, HRM analysis of 37 patients with known genotypes showed that all mutations were correctly detected Each genotype has its own unique melting curve. In a double-blind experiment, 28 abnormal melting curves were detected in HRM analysis of 70 unknown samples, which was consistent with the sequencing results. The sensitivity and specificity of HRM were 100%. Conclusion High-resolution melting curve analysis of HBB gene mutation detection is simple, rapid, sensitive and cost-effective. It not only can be used for mutation screening of β thalassemia, but also genotypes of known mutations.