Label-free fluorescent quantification of L858R single-nucleotide variant in EGFR gene in single-tube magnetic purification system.

The identification of single-nucleotide variations remains a significant challenge in genetic research due to their high similarity to wild-type DNA sequences. Many conventional approaches rely on sophisticated instrumentation or costly reagents, which limits their accessibility and broad application. To overcome these limitations, a novel detection strategy grounded in the well-established principle of restriction fragment length polymorphism (RFLP) was developed. In contrast to traditional electrophoresis-based RFLP methods, our approach employs biotin-labeled primers in conjunction with magnetic beads to enable efficient and instrument-free fragment separation. An additional advantage of this method lies in its label-free fluorescent detection, which reduces reagent costs and avoids signal interference commonly associated with fluorophore labeling. The system was validated by detecting the EGFR L858R mutation in genomic DNA from non-small cell lung cancer (NSCLC) patients. The method exhibited excellent linearity (r = -0.9995) and recovery rates between 85 % and 105 % in clinical sample analysis. The limit of detection for the L858R mutation ratio was determined to be 1.66 %, positioning the method comparably to commercial real-time PCR kits and next-generation sequencing (NGS) technologies. Compared to our earlier platform, this approach simplifies the workflow by eliminating probe washing and alleviating concerns about enzymatic inhibition caused by magnetic bead immobilization. Furthermore, this platform offers flexibility and can be readily adapted to detect a wide range of clinically relevant mutations through the selection of appropriate restriction enzymes. The simplicity of this method suggests significant potential for its application in point-of-care testing (POCT), particularly in clinical settings that are resource-limited or decentralized.