DON developments

NEW GRADING TECHNOLOGY AND NEW GENETICS BEING DEVELOPED IN ONTARIO

visual inspection of corn and wheat for Fusarium infection at the point of grain delivery is not accurate for determining actual levels of DON, so some grain is wrongly discounted at the expense of farmers. Laboratory-based methods for DON analysis (HPLC) and simple antibody-based ELISA tests work well, but are costly and take time and in some cases, complex equipment. That’s why Dr. Gregory Penner is developing a cost-effective handheld technology to rapidly determine levels of DON based on a different technology.

Current DON testing methods There are currently two types of DON testing: simple antibody-based ELISA tests and HPLC tests. ELISA (Enzyme Linked Immunosorbent Assay) tests take time and are complex, but provide reasonably accurate and cost-effective results. The ELISA technique uses the biochemistry of antigens and antibodies involved in the body’s immune response. When an antigen (which could be a toxin, bacteria or several other things) is introduced to a body, an antibody is produced that neutralizes the antigen by binding to it. The ELISA test involves the application of an antigen (in this case, the DON toxin, also known as deoxynivalenol) to a surface. An antibody is introduced, which binds with the antigen. The bound antibodies that remain are also linked to an enzyme, which is made to interact with a final introduced substance, and this interaction results in a chemical change that can be measured.   HPLC (High Performance Liquid Chromatography) tests are more complex, time-consuming and expensive, but provide a greater degree of accuracy. They involve the separation and identification of different compounds in a mixture using different stationary phases, a pump that moves sample material through a column, and a detector. “The primary difficulty with HPLC analysis is the cost of the equipment ($100,000) and the need for relatively sophisticated expertise in the use and maintenance of the equipment,” says Dr. Gregory Penner, co-owner of NeoVentures Biotechnology Inc. in London, ON. The Canadian Grain Commission currently charges $50 for an ELISA test and $195 for an HPLC test.   Other toxins such as aflatoxin and ochratoxin A are much more toxic than DON, so their tests require 1,000 times more sensitivity than those required for DON. Regulatory limits for toxins such as aflatoxin and ochratoxin A are in the parts per billion (ppb) range, whereas DON regulatory limits are in the parts per million (ppm) range.

“Antibodies are used in most detection kits for DON, and this is basically the same technology that is used in pregnancy tests,” says Penner, co-owner of NeoVentures Biotechnology Inc. in London. “Our innovation is to use DNA sequences (aptamers) that mimic antibodies.” He notes that when he and his partners started NeoVentures in 2002, they went through a process of identifying aspects of crop agriculture where new approaches could make a difference, “and it seemed that using aptamers for small molecules such as mycotoxins could provide the basis for a rapid, highly-sensitive test.”

However, the company team found that identifying aptamers for mycotoxins was only a starting point for commercial development, and figuring out how to use this technology to develop a testing device that could be sold in Canada and beyond was more difficult. “We’ve spent the last two years developing a rapid, high throughput detection technology for ochratoxin A (OTA),” says Penner. “To develop the OTA test as a commercial product we had to develop our own proprietary detection technology, but now that we have, we’re in a position to apply it to all mycotoxins. The purpose of this project is to enable us to adapt this technology for rapid, high throughput use with DON aptamers.”

how it works
In the NeoVentures approach, there are basically three steps involved. First, DON is extracted from the grain with an organic solvent. Then it is purified by passing it through a column that contains DNA sequences that bind and retain DON specifically. This enables a purification of DON from other grain components. DON is then released from the column and combined with a fluorescent signal and another aptamer in a tube. The amount of DON present can then be rapidly determined by measuring the amount of fluorescence. 

global recognition
During their work developing the OTA-Sense test, a call came in from Dr. Angelo Visconti and Dr. Annalisa De Girolamo at the leading mycotoxin research institute in the world in Bari, Italy. Penner and his team were delighted. “They are recognized world leaders and we are honoured that they believe we’re developing a platform that represents the next generation of mycotoxin testing,” Penner says. “Our collaboration with them is allowing us to access their grain samples and they are also providing highly accurate independent DON measurements for comparison.”

For this project, NeoVentures has hired Roberto Schena to work in Bari, in collaboration with three existing employees in London. NeoVentures is also collaborating with a German company on the development of a relatively inexpensive hand-held fluorometer.

using transgenes to fight disease
Ontario researchers are also making progress on the genetic side of managing Fusarium, the disease that causes DON in corn and wheat. Over the next two years, Dr. K. Peter Pauls will be evaluating the effectiveness of transgenes that better equip corn to fight Fusarium and Gibberella ear rot.

“Corn plants have a gene called rpl3, which produces a protein called RPL3,” says the University of Guelph Plant Agriculture professor. “Normal RPL3 proteins are inhibited by the DON mycotoxin, but initial tests showed that our transgenic lines of corn with a modified L3 (obtained from Steve Gleddie and Linda Harris at Agriculture and Agri-Food Canada in Ottawa) had disease scores 20 to 50 percent lower than normal plants after Fusarium inoculation.” Other lines of corn in the study have a modified Tri101 (trichothecene acetyltransferase) gene, which is able to convert the DON toxin produced by the fungus to a less toxic derivative. This fall, he will be able to analyze field data on the effectiveness of the rpl3 gene, with greenhouse and field testing of Tri101 transgenic plants to come next year. Pauls says it should be possible to someday combine both modes of disease defence action into one  transgenic plant. •