RPA-CRISPR/Cas12a-Mediated Isothermal Amplification for Rapid Detection of Phytopythium helicoides

Phytopythium helicoides, which belongs to algae (Chromista), Oomycota, Pythiales, Pythiaceae, and Phytophthora, is a quarantine pathogen that causes brown rot of fruits, stem rot and root rot, and other symptoms that can damage several tree species in urban landscaping. Therefore, disease management requires rapid and accurate diagnosis. The present study used recombinase polymerase amplification (RPA) in conjunction with the CRISPR/CRISPR-associated protein 12a (Cas12a) system to identify P. helicoides. The test exhibited high specificity and sensitivity and could detect 10 pg.μl of P. helicoides genomic DNA at 37°C within 20 min. The test results were visible by excitation of fluorophores by blue light. This groundbreaking test is able to detect P. helicoides in artificially inoculated rhododendron leaves. The RPA-CRISPR/Cas12a detection assay developed in this study is characterized by its sensitivity, efficiency, and convenience. Early detection and control of P. helicoides is crucial for the protection of urban green cover species.