Establishment of an RAA-CRISPR/Cas12a assay based on CpSge1 for rapid detection of Cryphonectria parasitica.

Chestnut blight disease caused by Cryphonectria parasitica is a serious branch disease that occurs worldwide, especially in Europe and North America. In recent years, chestnut blight disease has also been severe and even posed a great threat to the healthy development of chestnut orchards in some areas of China. Accurate and rapid detection of C. parasitica during the initial stages of the disease is helpful to take corresponding prevention and control measures in advance. In this study, we selected the CpSge1 (Gti1/Pac2 transcription factor family) of C. parasitica as the detection target and established a rapid and visual detection system of C. parasitica that combined the recombinase-aided amplification (RAA) and CRISPR/Cas12a, called CpSge1-RAA-CRISPR/Cas12a. The system allows for the specific detection of C. parasitica in approximately 60 mins, with visualization of results. The detection sensitivity of this system was found to be 1 pg/µL. We combined the RAA-CRISPR/Cas12a with a lateral flow dipstick, which also showed specific, high sensitivity, and fast characters. In conclusion, the RAA-CRISPR/Cas12a assay has great potential to be a method for early diagnosis and on-site detection of C. parasitica, especially for areas where specialized equipment is lacking.IMPORTANCEA rapid, highly sensitive, and visualized detection system of Cryphonectria parasitica was established by using the RAA-CRISPR/Cas12a method based on the C-terminal variable regions of a fungal-specific transcription factor CpSge1. The detection system was performed at a constant temperature condition of 37°C, which provides important support for the diagnosis of chestnut blight diseases in the field.