Low-Concentration Enhancement Effect: Enabling Sensitive Detection of EML4-ALK Variants via Multiplex RT-MIRA.

OBJECTIVE: ALK fusion variant detection is essential for guiding targeted therapy in non-small cell lung cancer (NSCLC). The emerging reverse transcription multi-enzyme isothermal rapid amplification (RT-MIRA) technology exhibits substantial potential in molecular diagnostics, enabling efficient variant profiling. However, its performance and optimization strategies for multiplex detection remain inadequately characterized. This study therefore aimed to analyze and optimize the RT-MIRA reaction system and develop a multiplex assay for detection of EML4-ALK variants (including V1, V2, and V3a/b variants) with high sensitivity. METHODS: Breaking from the conventional approaches, this study prioritized sensitivity as the primary evaluation metric during the development of the multiplex RT-MIRA assay, while ensuring the specificity of the reaction system. Reaction conditions were systematically optimized by combining time-to-threshold (TT) and relative fluorescence intensity (RFI) analyses. RESULTS: The optimized multiplex RT-MIRA assay achieved single-copy sensitivity for each variant plasmid. In a preliminary validation using 21 archived formalin-fixed paraffin-embedded (FFPE) clinical samples, the assay demonstrated complete concordance with commercial kits. Notably, we observed a "low-concentration enhancement effect," where reducing primer or probe concentrations within a specific range markedly improved detection sensitivity. CONCLUSION: By developing a systematic optimization strategy for multiplex RT-MIRA, this study provides a rapid and sensitive EML4-ALK detection tool and establishes a methodological foundation for broader clinical applications of RT-MIRA.