金纳米微粒DNA芯片检测EGFR基因突变

Gold nanoparticle-based detection of EGFR mutations on microarrays

目的:研制以金纳米微粒标记和银染色信号放大技术为基础的DNA芯片,用于快速检测表皮生长因子受体(EGFR)基因突变。方法:氨基修饰的寡核苷酸探针固定于醛基化玻璃片基制备DNA芯片,5′-生物素标记引物用于PCR扩增EGFR基因第18、19、20、21外显子片段,杂交后含互补序列的PCR产物结合于芯片,金纳米微粒借助表面包被的链亲和素识别标记于PCR产物5′-端的生物素而与之结合,再经银染色法放大杂交信号,依据芯片杂交结果判读EGFR基因突变,并用DNA直接测序法进行验证。结果:所制备的DNA芯片可快速检测肿瘤组织标本中EGFR基因突变,检测敏感性达到10-9 mol/L,可检出标本中5%的基因突变。结论:所制备的DNA芯片具有高特异性和敏感性,且操作简便,适用于临床肿瘤组织标本基因突变的快速检测。

Objective： To develop a DNA microarray based on gold nanoparticle and silver development for simple and rapid detection of mutations in epidermal growth factor receptor（EGFR） gene.Methods：The amino-modified probes that are designed to bind complementary DNA targets had been immobilized on the aldehyde-modified glass surface.Fragments containing the exon 18,19,20 or 21 of EGFR were amplified with PCR using 5’-biotinylated primers.The immobilized probes were used to capture complementary PCR amplicons,which were then detected by streptavidin coated gold nanoparticle.A silver development methodology was employed to amplify the signal and DNA sequencing was used to validate the genotype.Results：The EGFR mutations were detected rapidly using this DNA chip and gold nanoparticle-mediated silver staining.The lower limit of detection（LOD） of the assay was 10-9mol/L of PCR products.And it could distinguish 5% of mutation in sample.Conclusions：This gold nanoparticle-based microarray assay is able to detect and distinguish EGFR mutations.With advantages of high specificity and sensitivity and simpler procedure,this method can be used for quick detection of EGFR mutations in clinical work.