基于悬浮点阵技术的新型EGFR基因突变检测方法的建立和应用

Preliminary establishment and application of a new method for detection of EGFR mutations based on suspension array technology

目的构建基于悬浮点阵技术的新型表皮生长因子受体(EGFR)基因突变的检测方法,检测并分析非小细胞肺癌(NSCLC)组织中EGFR酪氨酸激酶抑制剂(TKI)相关的EGFR基因突变状况。方法选取61例经影像学和病理学检查确诊的NSCLC患者作为研究对象,手术后留取新鲜肺癌组织,提取基因组DNA,采用多重聚合酶链式反应(PCR)对组织样本的EGFR基因18、19、20和21号外显子中包含8种高频突变位点的特异片段进行扩增,并经多重连接酶检测反应(LDR)以特异性探针对扩增片段中的突变进行连接,再基于悬浮点阵技术平台对连接产物进行检测,最后通过DNA测序对检测结果进行验证。结果在61例NSCLC组织样本中,运用PCR-LDR联合悬浮点阵技术共检出EGFR基因突变19例,突变率为31.1%,其中外显子19缺失突变E746-A750 del(1)、E746-A750 del(2)各1例,外显子20点突变T790M 1例,外显子21点突变L858R 14例、L861Q2例,经DNA测序验证完全正确。结论运用PCR-LDR联合悬浮点阵技术初步建立了一种新型的EGFR基因突变检测方法,能同时准确检测EGFR基因中的8种高频突变,具有高通量和高特异性的特点,有望用于NSCLC患者进入EGFR-TKI药物靶向治疗前的EGFR基因突变检测。

Objective To explore a new method for detection of epidermal grouth factor receptor(EGFR) mutations based on suspension array technology,which can be used to detect and analyse EGFR mutations associated with EGFR tyrosine kinase inhibitors(TKI) in tissues of non-small cell lung cancer(NSCLC). Methods Sixty-one patients with NSCLC confirmed by imageology and pathology were selected,and the fresh tumor tissues were collected after operation for DNA extraction.The multiplex amplifications of exons 18-21 containing 8 EGFR mutations occurring in high frequency were performed by polymerase chain reaction(PCR),followed by ligation of the PCR products with a series of special probes using ligase detection reaction(LDR),then the LDR products were detected by suspension array technology platform.The results were compared with that from direct sequencing for confirming EGFR mutations. Results Nineteen EGFR mutations in 61 tissue samples of NSCLD(31.1%) were detected using multiplex PCR-LDR combined with suspension array technology,including 1 E746-A750 del(1) deletion mutation and 1 E746-A750 del(2) deletion mutation in exon 19,1 T790M point mutation in exon 20,and 14 L858R point mutation and 2 L861Q point mutation in exon 21.These EGFR mutations were verified by direct sequencing. Conclusion A new method for detection of EGFR mutations with high throughput and specificity has been established by PCR-LDR combined with suspension array technology,which can be used for accurate identification of 8 EGFR mutations occurring in high frequency simultaneously,and might be useful in detection of EGFR mutations for patients with NSCLC prior to targeted therapy with EGFR-TKI.