Objective Elaeocarpus spp. stem blight is a newly discovered branch disease on Elaeocarpus spp. caused by Pseudocryphonectria elaeocarpicola, for which research is still in its infancy and lacks effective prevention and control measures. This paper aims to establish a rapid and visualized method for the detection of P. elaeocarpicola, which provides an important technical support for the early monitoring and early warning of the disease.

Method In this study, we utilized PsGti1 gene of P. elaeocarpicola as a target, specifically amplified the target gene by recombinase aided amplification (RAA) reaction, and cut the target and fluorescent probe by CRISPR/Cas12a system, and finally visualized the detection of P. elaeocarpicola by lateral flow dipstick (LFD).

Result (1) In this study, the optimal primer pairs for the RAA amplification reaction of PsGti1 gene of P. elaeocarpicola were screened and obtained. (2) The CRISPR/Cas12a reaction system worked best when Cas12a and CrRNA concentration ratios were 1 and 0.125 μmol/L, respectively. (3) A RAA-CRISPR/Cas12a-LFD visualization detection system was established, which can realize the rapid detection of P. elaeocarpicola under the reaction condition of constant temperature at 37 ℃, and the sensitivity of detection was 50 fg/μL (gDNA as template) and 20 pg/μL (PsGti1_T-vector as template), respectively.

Conclusion The RAA-CRISPR/Cas12a-LFD detection system for P. elaeocarpicola established in this study has the advantages of easy-to-reach reaction temperature, high specificity, high sensitivity and ease of operation, which makes it suitable for carrying out the detection of P. elaeocarpicola in the field or in scenarios lacking laboratory testing equipment.