The pathogenic diseases are one important factor affecting rice growth, yield and quality, the development and application of rapid diagnostic methods targeting pathogens will contribute to the prevention and control of rice diseases. Herein, we developed one novel PAM-free loop mediated isothermal amplification assisted CRISPR/FnCas12a cleavage (Cas-PfLAMP) assays for the detection of three rice pathogens, such as Xanthomonas oryzae pv. Oryzae (XOO), rice stripe virus (RSV), and rice black-streaked dwarf virus (RBSDV). Furthermore, the Cas-PfLAMP on-spot detection system was also developed by combining solid phase nucleic acids extraction and lateral flow biosensor (LFB). The Cas-PfLAMP assay presented quite high specificity because of the double specific recognition from LAMP primers and FnCas12a/sgRNA, and high sensitivity as low as 10 aM. The results of inoculated rice leaves and practical field rice leaf samples confirmed the high performance of Cas-PfLAMP assay, indicating that the Cas-PfLAMP is one suitable on-spot detection method for rice diseases detection with high specificity, high sensitivity, and easy operation. We believe that the Cas-PfLAMP also could be extended to the other crop diseases detection, and other nucleic acid test fields as well.

PAM free loop mediated isothermal amplification coupled with CRISPR/FnCas12a cleavage for on-spot detection of three rice pathogens

ACS Spring 2022

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