CRISPR-Cas Detection Coupled with Isothermal Amplification of Bursaphelenchus xylophilus

Abstract

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 (gRNA) and CRISPR RNA (crRNA) were designed to target the 5S rDNA intergenic spacer sequences (IGS) region of B. xylophilus. Recombinase-aided amplification (RAA) was used for pre-amplification whose reaction condition was 37 oC for 15 min. The sensitivity of CRISPR/Cas12a could reach 94 copies/μl of plasmid DNA, or 2.33 copies/μl of purified genomic DNA (gDNA) within 45 min at 37 oC, while the sensitivity of CRISPR/Cas13a was 1000 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/100 mg of pine wood, 10-times higher than that of the CRISPR/Cas13a assay. This work enriches molecular detection approaches of 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.