Rapid and accurate detection of Fusarium oxysporum f. sp. Lycopersici using one-pot, one-step LAMP-CRISPR/Cas12b method

Together, our preliminary results illustrated that the LAMP-CRISPR/Cas12b method is a rapid simple, and reliable tool for Fol diagnosis and could be applied in point-of-need phytopathogen detection.

Here, we developed a novel method for Fol detection by integrating loop-mediated isothermal amplification (LAMP) assay and CRISPR/Cas12b detection in one-pot, and the whole reaction can simultaneously amplify and detect the target gene of Fol in one-step.

The total time of the present method is limited to 45 min and isothermally performed at 60°C. The limit of detection of this assay is 88.9 copies per reaction. The specificity of the LAMP-CRISPR/Cas12b method was 100% without any cross-reaction of other pathogens. A total of 24 nucleic acid samples were used to evaluate the performance of the LAMP-CRISPR/Cas12b method, including 12 with-Fol and 12 without-Fol. Compared with the gold standard results from real-time PCR, the present method provides a sensitivity of 100% (12/12), specificity of 100% (12/12), and consistency of 100% (24/24).