Ultrasensitive and on-site diagnosis of rice bakanae disease based on CRISPR-LbCas12a coupled with LAMP.

Abstract

Rice bakanae disease (RBD) has longstanding challenges impacted rice production, which is predominantly induced by Fusarium fujikuroi Nirenberg. Early diagnosis of F. fujikuroi is important to control RBD and improve quality and quantity of rice production. This study presents a novel on-site diagnosis platform combined with CRISPR/LbCas12a and LAMP to detect F. fujikuroi. LAMP amplification of TEF1-α, a characteristic gene of F. fujikuroi were performed, followed with trans-cleavage reaction of LbCas12a, cleaving the single-stranded DNA reporter, which is modified by the terminal fluorophore and quencher groups, producing fluorescence signal. The platform was confirmed with high specificity and sensitivity (LOD <1 aM). Furthermore, we designed a lateral flow strip experiment based on the trans-cleavage activity of LbCas12a, which was identified with similar sensitivity and specificity to the fluorescence detection method. In summary, this study achieved a platform with remarkable sensitivity and specificity for F. fujikuroi detection and provide potential for on-site and ultrasensitive diagnostic tools for RBD. © 2024 Society of Chemical Industry.