Validation of a Rapid, CRISPR-Cas-Free RPA-PCRD Strip Assay for On-Site Surveillance and Quarantine of Wheat Blast

As an emerging threat to global food security, wheat blast necessitates the development of a rapid and field-deployable detection system to facilitate early diagnosis, enable effective management, and prevent its further spread to new regions. In this study, we aimed to validate and improve an Recombinase Polymerase Amplification coupled with PCRD lateral flow detection (RPA-PCRD strip assay) kit for the rapid and specific identification of Magnaporthe oryzae pathotype Triticum (MoT) in field samples. The assay demonstrated exceptional sensitivity, detecting as low as 10 pg/µL of target DNA, and exhibited no cross-reactivity with M. oryzae Oryzae (MoO) isolates and other major fungal phytopathogens under the genera of Fusarium, Bipolaris, Colletotrichum and Botrydiplodia. The method successfully detected MoT in wheat leaves as early as 4 days post-infection (DPI) (asymptomatic plants), as well as in infected spikes, seeds, and alternate hosts. Furthermore, by combining a simplified polyethylene glycol-NaOH method for extracting DNA from plant samples, the entire RPA-PCRD strip assay enabled the detection of MoT within 30 min with no specialized equipment and high technical skills at ambient temperature (37-39 °C). When applied to field samples, it successfully detected MoT in naturally infected diseased wheat plants from seven different fields in wheat blast hotspot district, Meherpur in Bangladesh. This method offers a practical, low-cost, and portable point-of-care diagnostic tool suitable for on-site surveillance, integrated management, seed health testing, and quarantine screening of wheat blast in resource-limited settings. Furthermore, the RPA-PCRD platform serves as a modular diagnostic template that can be readily adapted to detect a wide array of phytopathogens by integrating target-specific genomic primers.