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

Chestnut blight disease caused by <i>Cryphonectria parasitica</i> 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 <i>C. parasitica</i> during the initial stages of the disease is helpful to take corresponding prevention and control measures in advance. In this study, we selected the <i>CpSge1</i> (Gti1/Pac2 transcription factor family) of <i>C. parasitica</i> as the detection target and established a rapid and visual detection system of <i>C. parasitica</i> that combined the recombinase-aided amplification (RAA) and CRISPR/Cas12a, called <i>CpSge1</i>-RAA-CRISPR/Cas12a. The system allows for the specific detection of <i>C. parasitica</i> 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 <i>C. parasitica</i>, especially for areas where specialized equipment is lacking.IMPORTANCEA rapid, highly sensitive, and visualized detection system of <i>Cryphonectria parasitica</i> was established by using the RAA-CRISPR/Cas12a method based on the C-terminal variable regions of a fungal-specific transcription factor <i>CpSge1</i>. 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.