Abstract
Single-nucleotide variation (SNV) is a crucial biomarker for drug resistance-related detection in cancer and bacterial infection. However, the unintended binding of DNA probes limits the specificity of SNV detection, and the need for redesigned sequences compromise the universality of SNV assay. Herein, we demonstrated a universal and low-cost assay for the colorimetric discrimination of drug-resistance related point mutation. By the use of a universal DNA probe and a split G-quadruplex, the signal could be recognized by naked eye at room temperature. The DNA probe was used as a signal reporter which not only improved the universality, but also enabled high specificity of probe hybridization. This assay was successfully applied in the detection of cancer-related SNV in the epidermal growth factor receptor (EGFR) gene, kirsten rat sarcoma viral oncogene homologue (KRAS), and tuberculosis drug-resistance related point mutation in RNA polymerase beta subunit gene (rpoB) with high specificity and visible readout. This method was simple, rapid, high-throughput and effective, which was suitable for point-of-care applications.
Highlights
Fluorescent modification is required and the format of nucleic acid detection is limited[24,25]
A highly sensitive and selective method for the detection of single-nucleotide variants (SNV) based on peptide nucleic acid (PNA) is reported[26,27,28]
We presented a universal strategy for the colorimetric detection of SNV with high specificity
Summary
Fluorescent modification is required and the format of nucleic acid detection is limited[24,25]. A highly sensitive and selective method for the detection of SNV based on peptide nucleic acid (PNA) is reported[26,27,28]. We presented a universal strategy for the colorimetric detection of SNV with high specificity. This assay was single-step, enzyme-free, non-labelled and low cost. It was based on a well-designed DNA probe (X-probe) with a split G-quadruplex as the signal reporter. The same A and B species could be used for kinds of X-probe designs and targeted different sequences All of these probes were unmodified which largely decreased the cost. The reaction mechanism of X-probe and its intended target (SNV) was based on the toehold-mediated strand displacement. The AP subspecies were displaced, and the G-quadruplex was split, resulting in the decrease of colorimetric signal
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