Highly Selective, Single-Tube Colorimetric Assay for Detection of Multiple Mutations in the Epidermal Growth Factor...

Many closed-tube methods are designed to detect DNA biomarkers. However, the utility of biomarkers such as a DNA mutation related to personalized medicine is limited as the operation of expensive detection instruments requires well-trained technicians. Therefore, we developed a simple and cheap colorimetric assay based on aggregation of silica-gold nanoparticle-modified probes, with linking probes, to detect mutations. This method consists of target amplification, sequence identification, and aggregation of the silica-gold nanoparticle-modified probes. All reactions are controlled by one individual and proceed sequentially, in a single tube, with no manual intervention. Approximately 10 copies of target DNA were detected with this assay, using 12 hot-spot mutations in exon 19 of EGFR gene as the example. In artificial samples, 0.1% mutant DNA can be distinguished from wild-type genomic DNA. The technology was tested on 104 clinical samples, which included 29 samples that were positive for an exon 19 deletion. The data were consistent with amplification refractory mutation system PCR, with the exception of one weakly positive sample, which was confirmed to be positive by digital PCR. The limit of detection of this colorimetric assay was verified to be better than that of amplification refractory mutation system PCR, and it provides a tool to discriminate multiple mutations in EGFR gene in clinical samples.