Rapid and Sensitive Detection of <i>Verticillium dahliae</i> from Complex Samples Using CRISPR/Cas12a Technology Combined with RPA

Verticillium wilt is primarily caused by the fungus <i>Verticillium dahliae</i> and represents one of the most important worldwide soilborne plant diseases. The causal agent can be spread by microsclerotia and conidia attached to seeds during national/international trade or in soil between fields. Consequently, accurate, sensitive, and rapid detection of <i>V. dahliae</i> from complex samples is critical for restricting entry of the pathogen to a new region/environment and enforcing early management of Verticillium wilt. Based on CRISPR/Cas12a and recombinant polymerase amplification (RPA) technologies, we developed an accurate, sensitive, and rapid detection method for <i>V. dahliae</i> with paper-based lateral flow strips. A highly efficient and specific CRISPR RNA (crRNA) was designed for the <i>GAPDH</i> gene of <i>V. dahliae</i> and was validated using several closely related <i>Verticillium</i> spp. Excluding the time required for the DNA extraction from the complex samples, a minimum of 40 min was required for the detection time. The RPA-CRISPR/Cas12a detection system had a lower detection limit of ∼10 copies of genomic DNA per reaction and was able to successfully detect as little as one microsclerotium per gram of soil. In addition, field samples displaying symptoms suggestive of <i>V. dahliae</i> were able to be positively identified for the presence of <i>V. dahliae</i>. Taken together, this study broadens the applications of CRISPR/Cas12a nucleic acid detection to soilborne crop diseases and will contribute to the future development of field-deployable diagnostic tools.