利用等位基因特异性扩增检测人结直肠癌BRAF基因V600E突变

Sensitive BRAF V600E mutation analysis of clinical samples using allele-specific amplification

目的建立一种基于等位基因特异性扩增技术检测人BRAF基因V600E突变的技术,用于检测低水平肿瘤点突变。方法设计选择性扩增人BRAF基因V600E的引物,利用BRAF V600E突变型大肠癌细胞系HT29核酸混合于BRAF野生型大肠癌细胞系SW480中进行灵敏度检测,通过与Sanger测序法比较,利用其检测40例结直肠癌石蜡标本的BRAF V600E突变情况。结果模拟细胞系混合检测显示该方法可检测出6.2%混杂于野生型基因里的BRAF V600E突变,利用该方法在40例大肠癌标本中成功检测出3例BRAF V600E突变,与测序法检出结果一致。结论成功建立了利用等位基因特异性扩增技术检测人BRAF基因V600E突变的实验技术并用于检测实体肿瘤标本,与测序法相比该方法具有灵敏、简便、快速的特点,可用于人肿瘤BRAF V600E突变的筛查应用。

Objective To evaluate the application of allele-specific amplification to detect low-lever BRAF V600E point mutation.Methods Allele-specific amplification and sequencing were used to detect the BRAF V600E point mutation in cell lines and 40 human colorectal tumors.The sensitivity of both assays were compared using serial dilutions of DNA extracted from HT29（BRAF V600E） cells in SW480（BRAF wild-type） cells.Results The detection limits of allele-specific amplification and sequencing were 6.2% and 12.5% respectively.3 colorectal tumors haboring BRAF V600E mutation could be detected with both methods.Conclusion Allele-specific amplification was rapid and sensitive in screening BRAF V600E mutation in colorectal tumors.