CRISPR-Cas Detection Coupled with Isothermal Amplification of Bursaphelenchus xylophilus

The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes significant damage to pine trees and, thus, poses a serious threat to pine forests worldwide, particularly in China, Korea, and Japan. A fast, affordable, and ultrasensitive detection of B. xylophilus is urgently needed for disease diagnosis. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have reshaped molecular diagnosis, with high speed, precision, specificity, strength, efficiency, and versatility. Herein, we established two isothermal diagnostics methods based on CRISPR-based platforms (CRISPR/Cas12a and CRISPR/Cas13a) for B. xylophilus-specific detection via fluorescence or lateral-flow strip readout. The guide RNA and CRISPR RNA were designed to target the 5S ribosomal DNA intergenic spacer sequences region of B. xylophilus. Recombinase-aided amplification was used for preamplification whose reaction condition was 37°C for 15 min. The sensitivity of CRISPR/Cas12a could reach 94 copies/µl of plasmid DNA, or 2.37 copies/µl of purified genomic DNA (gDNA) within 45 min at 37°C, while the sensitivity of CRISPR/Cas13a was 1,000 times higher than that of CRISPR/Cas12a of plasmid DNA in 15 min or 100 times higher of purified gDNA at the minimum reaction time of 4 min via fluorescence measurement. The CRISPR/Cas12a assay enabled the detection of 0.01 PWNs per 100 mg of pine wood, 10 times higher than that of the CRISPR/Cas13a assay. This work enriches molecular detection approaches for B. xylophilus and provides huge potential for ultrasensitive and rapid methods to detect B. xylophilus in pine wood, facilitating point-of-sample diagnostic processing for pine wilt disease management.