Rapid detection of Sclerotium rolfsii causing dry stem and root rot disease in cassava by recombinase polymerase amplification technique (RPA) combined with CRISPR/Cas12a

Abstract

Sclerotium rolfsii, a soil-borne phytofungus, is responsible for dry and stem root rot in cassava. PCR is a conventional molecular technique for living species identification but sophisticated equipment is required for DNA amplification. Herein, we developed recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a (RPA-CRISPR/Cas12a) to identify S. rolfsii. Optimal time for S. rolfsii detection by RPA-CRISPR/Cas12a performed at 37 °C was not exceeding 90 min. Detection limit of RPA-CRISPR/Cas12a for S. rolfsii in inoculated soil samples after three days was 1:1000 (g/kg) of fungi and planting soil, whereas conventional PCR was 1:10. Using the RPA-CRISPR/Cas12a assay to detect S. rolfsii in field stem samples showed significant differences when compared to conventional PCR. This indicated that the RPA-CRISPR/Cas12a system was superior to PCR because the RPA-CRISPR/Cas12a system detected S. rolfsii from both cassava stem and potting soil despite the absence of explicit signs of plant disease. Results demonstrated that the RPA-CRISPR/Cas12a assay was an effective technique for identifying pathogens, with potential for on-site testing.