Mr. Cole Reynolds Profile

Cole Reynolds

at Purdue Univ

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Proceedings Article | 13 June 2023 Presentation + Paper

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

Cole Reynolds, Xi Wu, Chutipol Thamaratnothai, Zheming Sun, Bartek Rajwa, Euiwon Bae, Martha Vaughan, J. Paul Robinson

Proceedings Volume 12545, 1254509 (2023) https://doi.org/10.1117/12.2665232

KEYWORDS: Lamps, Fluorescence, Microfluidics, Matrices, Portability, Fungi, Contamination, Agriculture

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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.

Proceedings Article | 1 June 2022 Presentation + Paper

Rapid molecular point-of-detection (POD) of mycotoxins-producing fungi in agricultural products using loop-mediated isothermal amplification (LAMP) assay

Xi Wu, Cole Reynolds, Euiwon Bae, Brianna Dowden, Yoo Hyun Kim, Bartek Rajwa, J. Paul Robinson

Proceedings Volume 12120, 121200B (2022) https://doi.org/10.1117/12.2623298

KEYWORDS: Agriculture, Fungi, Safety, Biological detection systems

Read Abstract +

Fungal species such as Aspergillus, Fusarium and Alternaria can contaminate agricultural commodities in the field or during storage and produce mycotoxins. They usually pose threats to human and animal health and can result in significant economic loss. Specifically, Fusarium graminearum, the major causative agent of Fusarium head blight (FHB) of small cereals produces mycotoxins including deoxynivalenol, nivalenol, and zearalenone. Conventional detection methods are time-consuming, expensive and require large-scale instruments and skilled technicians. Furthermore, detection of the toxins in post-harvested grain is a process that can only be accomplished after the grain is harvested. Therefore, our goal was to develop a molecular point-of-detection (POD) platform which was sensitive and specific to detect low levels of toxin-producing fungi within agricultural products in the field and could also be used directly in food products. Herein, we investigated a rapid molecular POD assay called loop mediated isothermal amplification (LAMP) to detect low levels of genomic DNA extracted from Fusarium graminearum, which is often associated with toxicological potential and food safety issues. Both fluorescent and colorimetric LAMP assays were characterized and optimized to detect low-level of pathogens within 70 and 50 minutes respectively. In summary, LAMP offers an efficient assay format for rapid and specific nucleic acid-based detection of mycotoxins in-field use. Coupled with our custom-designed microchip, our platform provides a proof-of-principle to achieve low-cost and widespread foodborne pathogens testing at the POD which is highly desirable to keep analysis time and costs low, but more importantly be a field use application.

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