高分辨率熔解曲线分析技术检测MASP-2基因点突变方法的建立及评价

Establishment and evaluation of detecting MASP-2 mutations by high-resolution melting analysis

摘要:

建立高分辨率熔解曲线（HRM）分析技术检测人甘露糖结合凝集素相关丝氨酸蛋白酶-2（ MASP-2）基因g.21370（G>T）位点单核苷酸多态性（SNP）的方法并进行评价。方法通过测序获得 MASP-2基因g.21370（G>T）位点3 种不同基因型对照品，并用其建立HRM 技术检测 MASP-2 基因点突变方法。利用60 份待测DNA 样品对建立的HRM 方法的准确性、重复性及稳定性进行评价。结果建立的 MASP-2 基因HRM 分析方法扩增反应正常，G/G、G/T、T/T 3种基因型区分明显，扩增产物Melt 曲线良好，无非特异性产物出现；与基因序列测定结果对比显示准确性良好；对野生型和突变型标本3 次重复检测结果显示，Tm 值变异系数为0.017 和0.023；χ2 检验结果表明，本研究选取的随机体检人群 MASP-2 基因g.21370（G>T）位点基因型频率分布符合Hardy-Weinberg遗传平衡。结论建立的 MASP-2 基因g.21370（G>T）位点HRM检测方法准确性高、重复性及稳定性良好，为下一步临床分析 MASP-2 基因SNP 提供一定的技术支持。

Abstract:

To establish a method for detection of mannose-binding lectin associated serine protease-2 ( MASP-2) mutations by high-resolution melting (HRM) analysis and to evaluate its effect. Methods Three different genotype reference substances of gene g.21370 (G>T) site were acquired by sequencing,and were used for establishment of HRM. Sixty DNA samples were used to evaluate the accuracy, repeatability and stability of this method. Results The amplification of gene analyzed by the HRM method was normal, three genotypes (G/G, G/T and T/T) were distinguished clearly, and the melting curves of the amplification products were good. The HRM analysis results were identical to the results of DNA sequencing.Repeated analyses showed that the coefficients of variance of Tm value were very small (0.017 and 0.023). x2 test showed that the distribution of genotype frequency of gene g.21370 (G>T) site in the selected population was accorded with Hardy-Weinberg genetic equilibrium. Conclusions The HRM method has high accuracy, good repeatability and stability for detection of gene g.21370 (G>T) mutations, whichprovides certain technical support for the clinical analysis of gene SNP in the next step.