One-pot RPA-CRISPR/Cas12a assay for rapid and sensitive detection of Sclerotium rolfsii in Glycine max.

Soil-borne fungal diseases are major constraints to the yield of legume crops, and it is therefore urgent to develop rapid and accurate detection methods for plant pathogens. In this study, a one-pot fluorescent detection system combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a was established for the rapid nucleic acid detection of Sclerotium rolfsii, the causal agent of soybean southern blight. The results demonstrated that the developed RPA-CRISPR/Cas12a one-pot assay could accurately identify the target pathogen within 45 minutes. The 95% limit of detection (LOD) was estimated to be 126.29 copies per reaction, and no cross-reactivity was observed with other common phytopathogenic fungi. The detection results of field soybean tissue and soil samples showed a consistency rate of 92.42% with the qPCR and full (100 %) agreement with ITS sequencing. Moreover, this assay was capable of detecting latently infected plants that could not be identified by routine field diagnosis, and qualified for pre-planting soil screening. This method is also applicable to the rapid and accurate screening of other phytopathogenic fungi in plant tissues and soil samples, and can be applied to the early warning and prevention of crop diseases.