Development of an RPA-based CRISPR/Cas12a assay in combination with a lateral flow strip for rapid detection of toxigenic Fusarium verticillioides in maize

Abstract

Fusarium verticillioides is an important phytopathogenic fungus that poses a threat to maize yield and quality in global maize-growing regions by causing Fusarium ear and stalk rot. The fungus is known to produce fumonisins, which are toxic secondary metabolites and have been associated with high incidences of esophageal cancer. The FUM1 gene is responsible for producing a crucial polyketide synthase required for fumonisin biosynthesis and is present in all pathogenic strains of F. verticillioides. This study aims to develop a rapid and accurate detection assay for F. verticillioides based on the FUM1 gene, by utilizing Chelex-100 resin for DNA extraction, recombinase polymerase amplification coupled with CRISPR/Cas12a cleavage and lateral flow detection (RPA-Cas12a-LFD) assay. The developed RPA-Cas12a-LFD assay exhibited remarkable specificity for F. verticillioides, and the lowest limit of detection was 2 ag DNA of F. verticillioides. The entire diagnostic process was completed in just 73 min, including sample DNA extraction, RPA reaction, Cas12a cleavage, and result readout. Furthermore, RPA-Cas12a-LFD assay was found to be equivalent to single-spore isolation and partial translation elongation factor 1α gene (TEF-1α) sequencing in identifying diseased samples in the field. In summary, this accurate and portable detection equipment has great potential for detecting and recognizing F. verticillioides, especially in areas where sophisticated lab equipment is not available.