Rapid detection of the pine wood nematode Bursaphelenchus xylophilus using recombinase polymerase amplification combined with CRISPR/Cas12a

Abstract

The pine wilt disease (PWD) caused by pine wood nematode (PWN), Bursaphelenchus xylophilus, is a destructive forest disease and causes global economic losses. Effective and affordable detection of PWN is critical for the management of this pest. In this study, we developed a rapid diagnostic assay using recombinase polymerase amplification (RPA) combined with the CRISPR/Cas12a system to detect PWN. The assay was validated as being fast, accurate, and reliable based on detection of PWN from pine wood samples. Test results for 33 PWN were positive, but negative for 11 isolates Bursaphelenchus species, 10 other nematodes, 20 isolates of 15 fungi species, and 6 isolates of Phytophthora species. This method allow us to have the results naked-eye directly. Results from this study indicated that this sensitive, specific, and rapid RPA-CRISPR/Cas12a assay has potentially significant applications to detect PWN, especially under time- and resource limited conditions.