Development and evaluation of a novel visual and rapid detection assay for toxigenic Fusarium graminearum in maize based on recombinase polymerase amplification and lateral flow analysis

Abstract

Maize ear rot caused by Fusarium graminearum is one of the most severe maize diseases in global maize-growing regions. It reduces maize yield in the field and is also responsible for mycotoxin contamination of grains during the postharvest period. F. graminearum is one of the major deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEN) producers. The ingestion of these mycotoxins represents a risk for human and animal health. Hence, early detection and identification of F. graminearum are crucial to controlling these mycotoxins along the food or feed supply chains. In this study, the recombinase polymerase amplification with lateral flow dipstick (RPA-LFD) assay targeting the gaoA gene that codes for galactose oxidase was developed. The reaction conditions were optimized to make the method rapid, sensitive, and cost-effective. The developed RPA-LFD assay could detect the presence of 20 fg of the target genomic DNA per reaction within 25 min at 40 °C. Moreover, 52 field samples were tested using the developed RPA-LFD assay and compared with conventional PCR-based methods. The positive rate between RPA-LFD and the conventional PCR-based method was 100%. In conclusion, the developed method provides a novel alternative for the rapid, sensitive, and specific detection and identification of F. graminearum. It is not only workable for bulk maize samples without using sophisticated lab equipment but is also potentially useful for other agriculturally important toxigenic fungi.