The Highly Selective Detection of Multiple Mutations of Exon 19 from Epidermal Growth Factor Receptor Gene in Single Tube Using the Colorimetric Assay

Abstract

Purpose: Many closed-tube methods were designed for detecting DNA biomarkers related to personalized medicine, but DNA biomarkers is difficultly detected because it need be operated by well-trained experimenters with expensive experiment facilities in a laboratory. Methods: To overcome this issue, a colorimetric assay based on the aggreggation of gold nanoparticle-modified probes with hairpin probe have been designed to develop a simple and low-cost method capable of detecting different mutation sites. This method was composed of three steps: target amplification, sequence identification and gold nanoparticle-modified probes aggreggation, and the steps were controlled by the temperature to proceed sequentially in one tube without any manual operation. Results: No equipment required, about 10 copies of target DNA, containing seven hot mutations at the exon 19 of EGFR gene, could be sensitively discriminated by using this assay, and 0.1% mutants of EGFR gene in artificial samples could be selectively distinguished from wild-type genomic DNA. Application on detecting 104 clinical samples, including twenty-nine 19del positive, show consistent results with ARMS-PCR, excluding a weakly positive sample.Conclusions: The colorimetric assay was verified to have a higher specificity than ARMS-PCR, and provides an available tool to discriminate different mutations at the exon 19 of EGFR gene in clinical samples.