High resolution melting curve analysis is a cost-effective rapid screening method for detection of somatic gene mutation. The performance characteristics of this technique has been explored previously, however, analytical parameters such as limit of detection of mutant allele fraction and total concentration of DNA, have not been addressed. The current study focuses on comparing the mutation detection efficiency of High-Resolution Melt Analysis (HRM) with Sanger Sequencing in somatic mutations of the EGFR gene in non-small cell lung cancer. The minor allele fraction of somatic mutations was titrated against total DNA concentration using Sanger sequencing and HRM to determine the limit of detection. The mutant and wildtype allele fractions were validated by multiplex allele-specific real-time PCR. Somatic mutation detection efficiency, for exons 19 & 21 of the EGFR gene, was compared in 116 formalin fixed paraffin embedded tumor tissues, after screening 275 tumor tissues by Sanger sequencing. The limit of detection of minor allele fraction of exon 19 mutation was 1% with sequencing, and 0.25% with HRM, whereas for exon 21 mutation, 0.25% MAF was detected using both methods. Multiplex allele-specific real-time PCR revealed that the wildtype DNA did not impede the amplification of mutant allele in mixed DNA assays. All mutation positive samples detected by Sanger sequencing, were also detected by HRM. About 28% cases in exon 19 and 40% in exon 21, detected as mutated in HRM, were not detected by sequencing. Overall, sensitivity and specificity of HRM were found to be 100 and 67% respectively, and the negative predictive value was 100%, while positive predictive value was 80%. The comparative series study suggests that HRM is a modest initial screening test for somatic mutation detection of EGFR, which must further be confirmed by Sanger sequencing. With the modification of annealing temperature of initial PCR, the limit of detection of Sanger sequencing can be improved.

中文翻译：

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EGFR的体细胞突变检测效率：高分辨率熔解分析与Sanger测序之间的比较。

高分辨率熔解曲线分析是检测体细胞基因突变的经济高效的快速筛选方法。先前已经探索了该技术的性能特征，但是尚未解决诸如突变等位基因部分的检测极限和DNA的总浓度之类的分析参数。本研究的重点是比较非小细胞肺癌中EGFR基因体细胞突变的高分辨率熔解分析（HRM）和Sanger测序的突变检测效率。使用Sanger测序和HRM相对于总DNA浓度对体细胞突变的次要等位基因部分进行滴定，以确定检测限。通过多重等位基因特异性实时PCR验证了突变体和野生型等位基因部分。体细胞突变检测效率 通过Sanger测序筛查275个肿瘤组织后，在116个福尔马林固定石蜡包埋的肿瘤组织中比较了EGFR基因的第19和21外显子。测序发现外显子19突变的次要等位基因部分的检出限为1％，HRM为0.25％，而外显子21突变的两种方法均检测到0.25％MAF。多重等位基因特异性实时PCR显示，在混合DNA分析中，野生型DNA不会阻碍突变等位基因的扩增。通过Sanger测序检测到的所有突变阳性样品，也通过HRM检测。通过测序未检测到在HRM中检测为突变的外显子19中约28％的病例和外显子21中40％的病例。总体而言，HRM的敏感性和特异性分别为100％和67％，阴性预测值为100％，阳性预测值为80％。对比系列研究表明，HRM是用于EGFR体细胞突变检测的适度初始筛选试验，必须通过Sanger测序进一步证实。通过修改初始PCR的退火温度，可以提高Sanger测序的检测限。