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.

由松材线虫(PWN)、松材线虫( Bursaphelenchus xylophilus )引起的松树枯萎病(PWD )是一种破坏性的森林病害，造成全球经济损失。有效且负担得起的 PWN 检测对于这种害虫的管理至关重要。在这项研究中，我们开发了一种使用重组酶聚合酶扩增 (RPA) 结合 CRISPR/Cas12a 系统来检测 PWN 的快速诊断方法。基于从松木样品中检测到 PWN，该测定被验证为快速、准确和可靠。33 种 PWN 的检测结果呈阳性，但 11 种伞滑刃属、10 种其他线虫、15 种真菌的 20 种分离株和疫霉属的 6 种分离株的检测结果呈阴性物种。这种方法可以让我们直接用肉眼看到结果。这项研究的结果表明，这种灵敏、特异和快速的 RPA-CRISPR/Cas12a 检测方法在检测 PWN 方面具有潜在的重要应用，尤其是在时间和资源有限的条件下。