OTA-Detect - A New Molecular based Method to detect Ochratoxin A producing fungi


Ochratoxin A (OTA) is a potent toxin mainly affecting the kidneys, causing both acute and chronic lesions, in mammals. OTA also displays hepatotoxic, genotoxic, teratogenic, and immunosuppressive properties and has been classified as a possible human carcinogen. In humans OTA has a half-life in blood of 35 days after oral ingestion and thus has been detected in human blood samples as well as in human breast milk. OTA is a common contaminant of cereals, grains, dried vine fruit, coffee and processed products from these sources including processed cereal foodstuffs and baby food. The risk to human health is through the intake of contaminated foods of vegetable origin and through foods of animal origin. Given the toxicity of OTA its presence in foodstuffs is clearly undesirable and there is increasing need by regulators and the food supply chain to provide better methods for analysis, detection and prevention of OTA and other mycotoxins. Many detection methods have been developed and commercialised by a range of specialist and general agri-food diagnostic companies ranging from chemical detection to immunoassays. Despite these developments, the market is continuing to require detection methods that can help with accuracy, speed and reduction of sample size and preparation issues. The OTA-Detect technology may be an option to overcome some of the current limitations by generating fast results with very small sample sizes as a pre-filter in the detection landscape.

Background
Mycotoxins are toxic metabolites produced by fungi. The FAO has estimated that up to 25% of the world's foods are significantly contaminated with mycotoxins. OTA is produced primarily by both Penicillium verrucosum and Aspergillus ochraceus but also by Aspergillus carbonarius, and Aspergillus alliaceus. OTA is a common contaminant of grains such as barley, corn, rye, wheat, and oats, with cereal-based products typically accounting for 50-80% of the average consumer intake of the toxin. It has also been reported in other plant products including coffee beans, spices, nuts, olives, grapes, beans, and figs. There is a risk to human health not only through the intake of contaminated foods of vegetable origin, but also through foods of animal origin with OTA being reported in animal derived food products, such as poultry and pork meat due to the feeding of mould contaminated feed to animals. OTA can survive many typical food-processing procedures, and has been reported in bread made from contaminated wheat and in wines and beers. There have been several recent reports of a high incidence (up to 54%) of OTA in human bloods. Recent European Union regulations have set maximum OTA levels of 0.5ppb for processed cereal-based foods for infants and young children, of 5.0 bbp for raw cereals and 3.0ppb for cereal products or cereals for direct consumption by humans and of 10ppb for dried vine fruit. In addition, sample size regulations require large (up to 10kg) samples be taken for OTA testing which in turn need to be processed for detection. This requirement is costly and inefficient.

Technology Description
The technology involves the use of a DNA based detection method which allows testing for the presence of OTA producing fungi in grains such as barley, maize and wheat and in a variety of cereal products. Samples which are positive for the fungus can subsequently be tested for the presence/activity of toxin production pathway enzymes, (using reverse-transcription (RT)- PCR) to determine if the fungi which are present in the cereals have produced OTA. The OTA-Detect technology uses PCR primers which are specific for the polyketide synthase (pks) gene which is essential for OTA production in these fungi. The method allows for the specific PCR based amplification of this gene from food samples, providing a means of testing for the presence of OTA-producing fungi. Having established the presence of OTA positive fungal DNA, an RT-PCR based approach can then be employed on these positive samples. If these genes are expressed then OTA has been produced. The RT-PCR based approach again uses the pks gene together with other OTA biosynthetic genes; to quantitatively determine the level of mRNA transcripts present, which correlates directly with the amount of OTA produced by the fungus in the sample

PCR detection of mycotoxigenic fungi


Correlation between OTA production and RT-PCR analysis of OTA gene in fungal strains grown in different conditions

Competitive Advantage and IP Status
The OTA-Detect technology has distinct advantages over the existing available methodologies:
1) Speed
2) Sensitivity/accuracy
3) Reduction in sample size (<1g)

Together these advantages provide the USP of a rapid 'pre-filter' diagnostic on sample feedstocks for subsequent chemical or immunoassay based detection of the toxin. This allows for multiple smaller samples to be taken from large batches if significant repetition of tests if required and helping greatly in the identification of localised mycotoxin contamination. A patent application has been filed for this technology, PCT (published) WO 2004/072224.

Type of Business Sought
This opportunity is available for discussion with potential licensees or other interested partners.

Principal Investigator
Professor Alan Dobson, UCC


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UCC is the owner of all IP

EI Bio works in partnership with UCC to commercialise the technology