| Summary: | <i>Phytophthora cambivora</i> is a major quarantine pathogen that devastates economically important plants across the globe. <i>P. cambivora</i> causes ink disease in chestnut trees and root and stem rot in various fruit trees, resulting in significant yield reductions and plant death. Given the potential dangers of <i>P. cambivora</i>, effective detection methods are needed for both disease management and prevention. In this study, based on the whole-genome screening of specific target genes, a combination of the recombinase polymerase amplification technique (RPA) and CRISPR/Cas12 was established to detect <i>P. cambivora</i>. The RPA-CRISPR/Cas12a assay was able to specifically detect 7 target isolates of <i>P. cambivora</i> but did not detect the following 68 non-target isolates, including 28 isolates of <i>Phytophthora</i>, 3 isolates of <i>Pythium</i>, 3 isolates of <i>Phytopythium</i>, 32 isolates of fungi, and 2 isolates of <i>Bursaphelenchus</i>. The RPA-CRISPR/Cas12a detection method was able to detect 10 pg·μL<sup>−1</sup> of <i>P. cambivora</i> genomic DNA at 37 °C within a short time span (60 min). Additionally, this method can identify the presence of <i>P. cambivora</i> in artificially inoculated apple fruits. In summary, compared with conventional detection techniques, the RPA-CRISPR/Cas12a detection method eliminates the need for expensive instruments, long reaction times, and high amounts of raw materials and can detect <i>P. cambivora</i> in imported plants at entry ports, enabling instant prevention and detection.
|
|---|