PAM-free loop-mediated isothermal amplification coupled with CRISPR/Cas12a cleavage (Cas-PfLAMP) for rapid detection of rice pathogens

Abstract

Pathogenic disease is an important factor affecting rice growth, yield and quality, and the development and application of rapid diagnostic methods will contribute to the prevention and control of rice disease. Herein, we developed a novel protospacer adjacent motif (PAM)-free loop-mediated isothermal amplification (LAMP) assisted CRISPR/Cas12a cleavage (Cas-PfLAMP) assay for detection of three rice pathogens; Xanthomonas oryzae pv. Oryzae (XOO), rice stripe virus (RSV), and rice black-streaked dwarf virus (RBSDV). The Cas-PfLAMP assay showed high specificity due to doubly specific recognition of LAMP primer sets and FnCas12a/sgRNA, and high sensitivity down to 9 or 3 copies due to LAMP amplification and CRISPR/Cas12a trans cleavage activity. Furthermore, a visual on-spot Cas-PfLAMP platform was established for detection of rice pathogens by combining solid-phase nucleic acid extraction and a lateral flow strip (LFS) test. Analysis of rice leaf field samples confirmed the impressive performance of the Cas-PfLAMP platform, demonstrating its suitability for rapid (∼50 min) on-spot detection of rice diseases. The assay could also be extended to detection of other plant diseases, and other nucleic acid field tests.

Keywords: CRISPR/Cas12a; CRISPR/Cas12a trans cleavage; Cas-PfLAMP assay; Lateral flow strip test; On-spot detection; PAM-Free LAMP; Plant disease; Rice pathogen; Solid-phase nucleic acid extraction.