Detection of mycotoxin-producing fungi in cereal crops using LAMP-based assays in a microfluidic device

Abstract

The consumption of mycotoxins generated by fungi can have severe effects on the health of both humans and animals. These toxins can exist at dangerous levels in food products made from crops that have been infected with mycotoxinproducing fungi. Numerous methods have been developed for detecting mycotoxins in order to divert contaminated commodities from the food supply, but only allow for reactive, not preventive approaches. Furthermore, under favorable conditions toxin-producing fungi can continue to produce mycotoxins during storage and throughout the crop processing stages. By identifying mycotoxin-producing fungal species on crops or commodities, remediation such as fungicide application can be carried out, preventing the spread of infection and potential contamination of healthy crops, reducing waste of resources and ultimately improving food safety. Loop-mediated isothermal amplification (LAMP) has advantages for portable DNA detection due to its isothermal nature, resistance to matrix inhibitors, and the possibility of a long shelflife when reagents are dried onto a matrix. The developed microfluidic device allows for the homogenized wheat sample input after DNA extraction. The microfluidic device functions as a disposable cassette and can be heated by an independent, portable, isothermal heating device. The LAMP assay is combined with calcein for fluorescence detection. In this experiment, Fusarium graminearum, a trichothecene mycotoxin producer, was used as a proof-of-concept for the device with a LAMP assay targeting the gaoA gene, which codes for the enzyme galactose oxidase (GO), a unique enzyme produced by only a few other fungal species. The presence of Fusarium graminearum was detected in contaminated wheat samples utilizing the described methods, indicating the potential detection of mycotoxin-producing fungi. In the future, the device will be expanded to test for multiple mycotoxin-producing genes.