RPA-CRISPR/Cas12a-Mediated Isothermal Amplification Technology for Visual Detection of Fusarium proliferatum

Abstract

Fusarium proliferatum is a fungal pathogen with an exceptionally broad host range, infecting ornamental plants and forest trees such as Populus and Cedrus deodara, and causing heart rot, root rot, and other diseases that lead to severe yield losses. It can also produce mycotoxins, including fumonisins, which threaten human and animal health. Conventional detection relies on isolation, culture, and morphological identification, which are time-consuming and prone to misidentification. Here, we established a rapid, visual molecular detection method by combining recombinase polymerase amplification (RPA) with the CRISPR/Cas12a system, targeting the F. proliferatum-specific gene Fpro_15953. The assay exhibited high specificity: the F. proliferatum isolate from C. deodara tested positive, while 37 non-target isolates (10 other Fusarium species, 10 other fungi, 15 oomycetes, and 2 Bursaphelenchus nematodes) produced no detectable signal. Under isothermal conditions at 37 °C, after 10 min of RPA followed by 10 min of Cas12a cleavage, as little as 0.001 ng·μL−1 of F. proliferatum genomic DNA could be detected. The method further succeeded in detecting F. proliferatum in artificially inoculated C. deodara needles and Populus × hopeiensis stem and root segments. This RPA-CRISPR/Cas12a platform provides an efficient, accurate tool for F. proliferatum detection and supports rapid field diagnosis.