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Literature sharing: Bursaphelenchus xylophilus detection by RAA-powered CRISPR-based diagnostics
Release date:
2026-03-12
Pine wilt disease (PWD) causes significant ecological and economic damage globally. Early detection of pine wood nematode (PWN), Bursaphelenchus xylophilus, the main causal agent of PWD, is critical for disease management.
Background
Pine wilt disease (PWD) causes significant ecological and economic damage globally. Early detection of pine wood nematode (PWN), Bursaphelenchus xylophilus, the main causal agent of PWD, is critical for disease management.
Limitations of current detection methods
Traditional morphological methods: time-consuming and error-prone.
PCR-based methods: involve complex procedures, require relatively long time and expensive equipment.
Isothermal amplification-based methods (e.g., LAMP and RPA): prone to nonspecific amplification, and the design of primers and probes is too complex for nonexperts.
Considering CRISPR-based diagnostics (CRISPR-Dx) offer high sensitivity, specificity, and speed, this study aims to develop CRISPR/Cas12a and CRISPR/Cas13a assays for B. xylophilus detection.
Results
Establishments of CRISPR-Dx diagnostics for B. xylophilus
A species-specific sequence from B. xylophilus 5S rDNA IGS was selected as target sequence for detection. A gRNA was designed based on PAM sites for CRISPR/Cas12a detection (Fig. 1A) and the sensitivity was 100 ng/µL (Fig. 1B). crRNA1 was designed for the CRISPR/Cas13a platform (Fig. 1A) which yielded a detection sensitivity of 100 pg/µL (Fig. 1C). These results validated the feasibility of the CRISPR-Dx systems in B. xylophilus detection.
Optimization of CRISPR-Dx diagnostics platform
To further enhance diagnostic accuracy, a preamplification step was applied before CRISPR-Dx detection using RAA kits (Qitian). Four pairs of RAA primers (RAA-5S-F1/R1 to RAA-5S-F4/R4; RAA-5S-F1/R1 to RAA-5S-F3/R3 were shown in Fig. 1A) were paired into 16 sets. The best performing primer set was selected according to the fluorescent signal produced by the CRISPR/Cas12a or CRISPR/Cas13a platform (F1 + R2 for both platforms). The optimal primer concentration was 600 nM for both platforms.
Specificity of CRISPR-Dx diagnostics between B. xylophilus and B. mucronatus
The specificity of the assay was evaluated using gDNA extracted from B. xylophilus and B. mucronatus (Fig. 3). No fluorescent signal cross-reactivity was observed in either CRISPR/Cas12a or CRISPR/Cas13a assays, and positive reactions were only obtained with gDNA from B. xylophilus, indicating that the CRISPR-Dx methods are specific for B. xylophilus. The result of the lateral-flow strip analyses further supported this conclusion.
Sensitivity of CRISPR-Dx diagnostics
After coupling with RAA, the analytical sensitivity of CRISPR/Cas12a could reach 94 copies/µL of plasmid B. xylophilus DNA (the LOD for ddPCR), or 2.37 copies/µL of purified gDNA within 45 min at 37 °C (which could not be detected by ddPCR; Fig. 4–5). The sensitivity of CRISPR/Cas13a was 1,000-fold higher than that of CRISPR/Cas12a for plasmid DNA in 15 min and 100-fold higher for gDNA at the minimum reaction time of 4 min via fluorescence measurement (Fig. 4–5). However, the use of paper-strip lateral-flow readout does not provide the same sensitivity as fluorescence-based detection. The LOD for lateral-flow was 7.7 × 10³ copies/µL for plasmid DNA and 2.93 × 10⁴ copies/µL for gDNA for both platforms.
Application and performance of CRISPR-Dx diagnostic for pine wood samples
In simulated pine wood samples, the sensitivity of the CRISPR/Cas12a assay was 0.01 PWN in 100 mg of simulated wood samples after 60 min, while the CRISPR/Cas13a could detect a minimum of 0.1 PWN per 100 mg of pine wood after 20 min. CRISPR-Dx diagnostics methods are more sensitive than the RPA assay (1 PWN per 100 mg of pine wood within 25 min), which is reportedly the most sensitive molecular diagnostic technology available for B. xylophilus detection.
Conclusion
This study successfully established RAA-coupled CRISPR/Cas12a and CRISPR/Cas13a assays for rapid, specific, and ultrasensitive detection of B. xylophilus. These methods have complementary advantages: CRISPR/Cas12a directly targets DNA with simpler operation, while CRISPR/Cas13a offers higher sensitivity and potential advantages for detecting live pathogens.
These methods represent promising tools for field diagnosis and quarantine management of pine wilt disease.
Reference
Tang C, Wu J, Chen Q, Wang Y. CRISPR-Cas Detection Coupled with Isothermal Amplification of Bursaphelenchus xylophilus. Plant Dis. 2023 Jun;107(6):1703–1713. doi: 10.1094/PDIS-07-22-1648-SR.
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