Rapid and sensitive detection of Fusarium graminearum in maize using an RPA–CRISPR/Cas12a system

Fusarium graminearum is a destructive fungal pathogen that causes major diseases in cereal crops like maize, wheat, and barley. In maize, it is a primary causal agent of stalk rot, leading to significant yield losses and contaminated grain with mycotoxin that threaten human and animal health. This study presents and evaluates a novel nucleic acid detection platform that combines recombinase polymerase amplification (RPA) with a CRISPR/Cas12a system for the rapid identification of F. graminearum in maize. By targeting the translation elongation factor 1α ( EF-1α ) gene, the assay discriminates F. graminearum from related species with high conservation. Following systematic optimization, the proposed method exhibited high sensitivity and specificity for the detection of F. graminearum using both lateral flow strips (LFS) and green fluorescence visualization. The method enabled the detection of F. gra min earum DNA at concentrations as low as 0.63 pg (13 copies) within 20 min, while it reliably identified infections in maize coleoptiles and field samples as early as 4 days post-inoculation. Notably, this approach provides a novel alternative for the rapid, sensitive, and specific visualization, detection, and identification of F. graminearum without requiring specialized technical expertise or costly instrumentation. By integrating CRISPR/Cas12a specificity with the rapid amplification capability of RPA, this assay represents a powerful tool for early and accurate pathogen detection in maize production systems.