Double-probe rolling circle amplification with lateral flow readout for highly specific detection of the phytopathogenic and keratocutaneous pathogen Fusarium proliferatum

Fusarium proliferatum is a soilborne phytopathogenic fungus that poses a serious threat to global crop production owing to its broad host range, strong toxigenicity, and wide distribution. Beyond its agricultural impact, this fungus has also emerged as an opportunistic pathogen capable of infecting human cornea and skin, particularly in immunocompromised individuals. Rapid and accurate on-site detection is critical for early disease intervention. In this study, we adapted and optimized a dual-probe rolling circle amplification integrated with lateral flow assay (dpRCA-LFA) for the specific detection of F. proliferatum , building on previously reported dpRCA-LFA principles. The main contributions of this work are the design of F. proliferatum -specific probes and the optimization of the reaction system for soil and field sample testing. The conventional single padlock probe was redesigned into two probes (long probe and short probe), establishing a dual-recognition mechanism that substantially enhances specificity. Under optimized conditions, the lowest concentration at which a visible positive signal was observed was 6.6 × 10 −4 ng μL −1 for genomic DNA and 2.7 × 10 2 copies μL −1 for recombinant plasmid containing the TEF-1α. These values represent the detection end-points under the tested conditions rather than statistically established limits of detection, and correspond to a 100-fold improvement over conventional PCR-AGE in the tested dilution series. Specificity testing against 19 fungal strains yielded positive signals exclusively for F. proliferatum . The assay exhibited robust anti-interference performance, with consistent detection even when target DNA constituted only 1% of total DNA. In artificially inoculated soil samples, dpRCA–LFA achieved a 10-fold higher sensitivity than PCR–AGE. In field samples, dpRCA-LFA identified five positive samples versus four detected by PCR-AGE. Given the limited number of field samples, this difference is presented as a preliminary observation rather than a demonstration of superior field performance. The entire process, from amplification to visual result interpretation, can be completed within 90 min without requiring specialized equipment. Collectively, dpRCA-LFA offers a rapid, sensitive, and specific tool for the detection of F. proliferatum in fungal cultures and soil matrices, with potential utility for decentralized screening after suitable DNA extraction and controlled reaction processing. Clinical application remains a future direction.