LAMP-Based Two-DNA-Fragment Fusion and Its Application in Nucleic Acid Detection.

Loop-mediated isothermal amplification (LAMP) continuously generates strand-displaced single-stranded DNA intermediates, providing the possibility of assembling DNA fragments. Here, we developed a novel two-DNA-fragment fusion technique, termed fusion LAMP, which is an isothermal DNA-fusion strategy that enables the fusion of two independent DNA fragments within a single amplification reaction. By combining fusion LAMP with CRISPR/Cas13a, we further established an "AND-gate" nucleic acid detection platform, termed Fusion LAMP-Coupled CRISPR/Cas13a (FLCC), which enables concurrent detection of two targets by reading the fusion product-triggered fluorescence signals. This platform generates signals only when two targets are present simultaneously. To prove this concept, we then employed FLCC to identify the methicillin-resistant Staphylococcus aureus (MRSA). This method achieved a limit of detection of 10 copies/μL of MRSA genomic DNA and showed no cross-reactivity with closely related bacterial strains. Furthermore, we validated its feasibility by detecting 19 clinical isolates, demonstrating a simple and accurate approach for MRSA detection. Collectively, the FLCC platform ensures identifying pathogens accurately and provides a promising diagnostic approach for detecting complex genetic targets.