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Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a
Published on: December 23, 2022
PAM-free loop-mediated isothermal amplification coupled with CRISPR/Cas12a cleavage (Cas-PfLAMP) for rapid detection
Zaobing Zhu1, Rong Li1, Hanwen Zhang1
1National Center for the Molecular Characterization of Genetically Modified Organisms, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
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.
