Development of an RPA-CRISPR/Cas12a Assay for Rapid and Sensitive Diagnosis of Plant Quarantine Fungus <i>Setophoma terrestris</i>

Abstract

Setophoma terrestris is an important phytopathogenic fungus listed by China as a harmful fungus subject to phytosanitary import control. This pathogen is a threat to a wide range of plants, particularly as the causal agent of onion pink root rot, one of the most severe diseases of onions. In order to provide rapid identification and early warning of S. terrestris and prevent its spread, we have developed a rapid, accurate, and visually intuitive diagnostic assay for this pathogen, by utilizing recombinase polymerase amplification (RPA), coupled with CRISPR/Cas12a cleavage and fluorescence-based detection systems or paper-based lateral flow strips. The developed RPA-CRISPR/Cas12a assay exhibited remarkable specificity for the detection of S. terrestris. Moreover, this protocol can detect the pathogen at a sensitivity level of 0.01 pg/μL, which significantly outperforms the 1 pg/μL sensitivity achieved by the existing qPCR-based detection method. The entire diagnostic procedure, including DNA extraction, the RPA reaction, the Cas12a cleavage, and the result interpretation, can be accomplished in 40 min. Furthermore, the successful application of the assay in infected plant samples highlighted its potential for rapid and accurate pathogen detection in agricultural settings. In summary, this RPA-CRISPR/Cas12a diagnostic method offers a potentially valuable technological solution for quarantine and disease management.

Keywords

• fluorescence detection
• lateral flow detection
• onion pink root rot
• phytosanitary pathogenic fungi
• RPA-CRISPR/Cas12a
• visual detection