Developing rapid tests for mycotoxins

Story written by Bruce Barker | The Canadian Agronomist

Knowledge is power. That’s the rational for a research project partially funded by Alberta Grains that is looking at how to develop a rapid test that can detect deoxynivalenol (DON) levels in cereals grains in five minutes and costs less than five dollars per sample. And with that knowledge, farmers will have the power to better manage Fusarium head blight infestations and grain marketing strategies.

“One thing I would say, the technology we're developing is never going to replace some of the really expensive analytical tools that grain elevators and food processors need to do, because when you really need to know what the level of contamination is, you're going to need that really high tech equipment,” says Maria DeRosa, Dean of the Faculty of Science at Carleton University, Ottawa, ON. “But what we're hoping is that high throughput screening with quick, multiple samples could be done in a really cheap but effective way so it could save money in the long run.”

The mycotoxins that DeRosa is developing rapid tests for are DON, Ochratoxin A (OTA), Fumonisin B1 (FB1) and Aflatoxin B1 (AFB1). Of these, DON is of most interest in western Canada where the fungal pathogen that causes Fusarium head blight in cereals can result in the production of DON in the seed. While DON is a relatively mild toxin, it can cause issues when feeding to livestock, and can also have human health impacts.

The EU has set the maximum permitted level for DON in cereal-based foods for infants and children at 0.20 ppm, as well as cereals intended for direct human use at 0.75 ppm, and finished wheat products for human consumption at 1.00 ppm. The U.S. Food and Drug Administration (FDA) has also established guidance or action levels for DON in unprocessed and processed foods, which range from 1 ppm to 10 ppm depending on the use. In Canada, Health Canada has established maximum DON levels at 1 ppm for grain fed to swine, young calves and lactating dairy animals, at 2 ppm for uncleaned soft wheat for human consumption, and at 5 ppm for cattle and poultry diets.

Currently, the only way to measure DON content is to send a grain sample to the Canadian Grain Commission or other reputable laboratory. An ELISA tests for DON can cost over $20 a sample and take two days for results. DON analysis by LC-MS/MS can cost $60 and take up to five days for results. As a result, rapid test kits capable of DON detection at the sub ppm level could be very useful for reliably screening samples at a low cost says DeRosa.

DeRosa’s research on mycotoxin detection goes back 15 years in the medical field where she hoped to be able to rapidly and cheaply detect things like cancer cells or bacterial infections. A colleague at Carleton University, Professor David Miller, introduced her to the field of mycotoxins in agriculture where he saw a need for a rapid and economical test.

The technology DeRosa is using is based on ‘aptamers’, which are synthetic RNA or DNA strands that bind with high specificity to target molecules. DeRosa’s lab has identified aptamers that target DON, OTA, FB1, and AFB1, and has begun to apply these aptamers to develop rapid, low cost mycotoxin tests.

“So these are pieces of DNA that can interact with the different toxins in a specific way. Finding those sequences, optimizing them, and now in this latest phase, putting them in a platform that is easy to use and super cheap is our next step,” she says.

As a first step, DeRosa and her colleagues Dr. Velu Ranganathan and Dr. Sathya Srinivasan worked on integrating the aptamer technology with the established lateral flow assay (LFA) technique. The LFA provides a test strip platform for the aptamer technology. She likens the technology to a quick Covid antigen test. In the Covid test, a negative test will show a colored ‘C’ line when exposed to a collected sample. A positive test will have a colored line at both the ‘C’ and ‘T’ line.

The initial research worked with the OTA mycotoxin to improve the test strip reliability and accuracy on wheat and corn samples that were obtained from collaborators at Agriculture and Agri-Food Canada (AAFC), research scientist Dr. Mark Sumarah and his technician Megan Kelman.

With proven success with OTA lateral flow test strips, DeRosa moved on to developing a similar test for DON. After refining the DON test strips, detection levels of approximately 13 ppb were achieved. The test strips were then evaluated with naturally occurring DON extracts from corn and wheat obtained from AAFC. With these naturally occurring samples, detection was in the range of 4 ppm.

A small plot field trial was also conducted to assess the test strips. Seven wheat and corn samples from Agriculture and Agri-Food Canada were analyzed for DON and 13 of 14 showed good results with detection levels well below 2 ppm.

The research also looked into developing a multiplexed test strip that could detect the presence of FB1, OTA, DON, and AFB1 with one test strip. Further refinement of a colour-based test strip is required before field testing.

An area of refinement that is needed to move from the lab to the field is the method used to extract the sample for use with the test strips. DeRosa currently uses multiple steps and specialized equipment. Ideally, a field extraction method would use water.

“Maybe it's a filter like was used with a COVID test. You take the sample, squeeze it through a filter, and drops come out. You put the drops on the test strip. I think it has to end up looking no more complicated than that,” says DeRosa.

DeRosa also hopes to be able to quantify the results that shows a specific level of mycotoxin rather than just a positive number at a predetermined level. Because the line on the test strip fades differently with different concentration levels of the mycotoxin, there is an opportunity to quantify the concentration level.

“If someone has a backyard pool, you have test strip to dip in the water and compare it to a colour strip to measure pH or chlorine levels. That gives a rough idea of how it could work, but the future is moving more toward using a cell phone with some kind of reader to get as quantitative as possible,” says DeRosa.

Farmers could benefit from the technology in several ways. Actually knowing what DON levels are on a field to field basis could help with grain storage allowing a farmer to segregate grain based on DON levels. Or they could blend grain with higher DON levels with low or non-existent levels to lower the DON levels if DON content ever became a grading feature with the Canadian Grain Commission.

The rapid DON test could also help farmers track Fusarium head blight infestations through the years and on individual fields. This knowledge could help with future management decisions to minimize the disease that could include altering crop rotations, selecting varieties with the highest level of resistance, increasing seeding rate, and using seed treatments to control seed-borne pathogens.

“Our main goal is to support the whole pipeline from farm to grain elevator to food processor. Those are the people that we think could benefit the most from being able to make more informed decisions on what they're doing with their grains,” says DeRosa.

Overall, the research funded by Alberta Grains, Agriculture Development Fund (Govt of Canada and Saskatchewan), Western Grains Research Foundation, Manitoba Crop Alliance and the Saskatchewan Wheat Development Commission will give farmers more power over Fusarium head blight.

“We got some really exciting results. It looks like in several of the cases, not only does the technology work, but it is actually even in the right ballpark to make regulatory decisions,” says DeRosa. “The fact that we got to this point is really encouraging, and now sets us up to try to get this into a true field test where we would be completely outside of the lab with real samples in the hands of folks who were going to have to make these decisions.”