Rapid Detection of Phytophthora cambivora Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12a

Abstract

Phytophthora cambivora is a major quarantine pathogen that devastates economically important plants across the globe. P. cambivora 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 P. cambivora, 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 P. cambivora. The RPA-CRISPR/Cas12a assay was able to specifically detect 7 target isolates of P. cambivora but did not detect the following 68 non-target isolates, including 28 isolates of Phytophthora, 3 isolates of Pythium, 3 isolates of Phytopythium, 32 isolates of fungi, and 2 isolates of Bursaphelenchus. The RPA-CRISPR/Cas12a detection method was able to detect 10 pg·μL−1 of P. cambivora genomic DNA at 37 °C within a short time span (60 min). Additionally, this method can identify the presence of P. cambivora 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 P. cambivora in imported plants at entry ports, enabling instant prevention and detection.

Rapid Visual Detection of Peronophythora litchii on Lychees Using Recombinase Polymerase Amplification Combined with Lateral Flow Assay Based on the Unique Target Gene Pl_101565

“…Typically, the RPA reaction can be performed within 20 min at temperatures between 22 and 45 • C [16]. To determine the optimal temperature for the established RPA-LF assay, eight different incubation temperatures (25,28,31,34,37,40,43, and 46 • C) were evaluated, and 100 ng of P. litchii genomic DNA were used as a template. To determine the optimal detection time, RPA reactions were carried out at 37 • C for times of 5, 10, 15, 20, 25, 30, and 35 min, with each assay using 100 ng of genomic DNA as a template.…”