Development of an RPA-based CRISPR/Cas12a assay in combination with a lateral flow strip for rapid detection of toxigenic Fusarium verticillioides in maize
[en] 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.
Disciplines :
Agriculture & agronomy
Author, co-author :
Liang, Xiaoyan ; Université de Liège - ULiège > TERRA Research Centre ; Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences/Key Laboratory of Agro-products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs, Beijing, China
Zhang, Xiu; North Minzu University, Ningxia Key Laboratory for the Development and Application of Microbial Resources in Extreme Environments, Yinchuan, China
Xi, Kaifei; Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences/Key Laboratory of Agro-products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs, Beijing, China
Liu, Yang; School of Food Science and Engineering, Foshan University/National Technical Center (Foshan) for Quality Control of Famous and Special Agricultural Products (CAQS-GAP-KZZX043)/Guangdong Key Laboratory of Food Intelligent Manufacturing, Foshan, China
Jijakli, Haissam ; Université de Liège - ULiège > Département GxABT > Gestion durable des bio-agresseurs
Guo, Wei ; Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences/Key Laboratory of Agro-products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs, Beijing, China
Language :
English
Title :
Development of an RPA-based CRISPR/Cas12a assay in combination with a lateral flow strip for rapid detection of toxigenic Fusarium verticillioides in maize
NSCF - National Natural Science Foundation of China
Funding text :
This work was supported by the National Key Research and Development Program of China ( 2022YFE0139500 and 2022YFD1400100 ), the National Natural Science Foundation of China (No. 32072377 ), Agricultural Science and Technology Innovation Program of Institute of Food Science and Technology , Chinese Academy of Agricultural Sciences ( CAAS-ASTIP-G2022-IFST-01 ), Central Public-interest Scientific Institution Basal Research Fund ( Y2022GH15 ), Science and Technology Leading Talents of Ningxia Hui Autonomous Region ( 2022GKLRLX06 ), and Open Foundation of Ningxia Key Laboratory for the Development and Application of Microbial Resources in Extreme Environments ( NXTS04 ).
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