Rapid and sensitive detection of Verticillium dahliae from complex samples using CRISPR/Cas12a technology combined with RPA.

Verticillium wilt is primarily caused by the fungus Verticillium dahliae, 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 V. dahliae from complex samples is critical for restricting entry of the pathogen to a new region/environment and 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 V. dahliae with paper-based lateral flow strips (PLFS). A highly efficient and specific CRISPR RNA (crRNA) was designed for the GAPDH gene of V. dahliae, and was validated using several closely-related Verticillium 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 V. dahliae were able to be positively identified for the presence of V. dahliae. 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.