A one-pot RPA/CRISPR-bio-dCas9 lateral flow assay for rapid and on-site detection of Monilinia fructicola in stone and pome fruits.

Abstract

BACKGROUND Brown rot, a devastating fungal disease affecting stone and pome fruits, leads to substantial economic losses worldwide, impacting production, post-harvest storage, and transportation. Monilinia fructicola, the primary causal agent of brown rot, is especially challenging to manage because of its ability to establish latent infections in fruit, making early detection and control extremely difficult, enabling the disease to spread unnoticed. Therefore, it is essential to develop fast and accurate detection technologies. RESULTS In this study, a one-pot detection method for M. fructicola, integrating CRISPR-bio-dCas9, recombinase polymerase amplification (RPA), and a lateral flow assay (LFA) was developed. This method could detect the M. fruticola genome in less than 30 min from sample collection to result, with a detection limit of 4 copies/μL of M. fructicola. The RPA/CRISPR-bio-dCas9 LFA method simplified M. fructicola detection by eliminating the need for probes, additional reporters, or specialized equipment, thereby reducing costs and complexity. Furthermore, the streamlined single-tube workflow minimized cross-contamination risks, enabling non-expert workers to perform efficient pathogen screening. CONCLUSIONS The RPA/CRISPR-bio-dCas9 LFA enabled advanced brown rot management by addressing the limitations of traditional detection (slow speed, high cost, complexity). With high sensitivity and a short detection time using an equipment-free design, it enabled practical on-site detection of M. fructicola, allowing timely interventions to reduce pre- and post-harvest losses in fruit production. © 2025 Society of Chemical Industry.