Characterization of Recombinase Polymerase Amplification Primers Targeting EGFR Exon 19 Deletions for a Proposed Isothermal Lung Cancer Diagnostic Workflow

The analysis of circulating tumor DNA (ctDNA) holds significant promise for early lung cancer detection. However, current genomic screening modalities remain limited by laboratory costs and resource access. This study presents the preliminary design and experimental characterization of Recombinase Polymerase Amplification (RPA) primers designed to target the EGFR exon 19 deletion (ex19del) biomarker using synthetic control templates. This primer framework is evaluated as the foundational core for a proposed downstream workflow intended to eventually incorporate CRISPR-Cas9 specificity processing and Lateral Flow Assay (LFA) readouts. In vitro testing demonstrated successful amplification of the target sequence, with a 5 bp overlap forward primer yielding prominent amplification. Concurrently, persistent non-specific amplification was observed, establishing the necessity for subsequent enzymatic filtration. While the complete LFA readout and CRISPR-Cas9 components represent future development steps that require clinical verification in plasma samples, this work validates the operational range of the foundational amplification primers.