Développement de méthodes bioanalytiques à base d'aptamères pour la determination de l'ochratoxine A

Abstract

Ochratoxin A (OTA), an isoucoumarinic mycotoxin produced by the genra Aspergillus and Penicillium, is a common contaminant of several foodstuffs. Even in trace amounts, OTA has adverse affects on human and animal health: nephrotoxic, teratogenic, neurotoxic, immunosuppressive and it was considered by the international Agency of Research on Cancer (IARC) as a potential carcinogenic agent (group 2B). In order to meet food safety concerns and official legislated regulations, analytical methods, mainly based on chromatographic techniques, have been reported for OTA detection. Despite their effectiveness, these techniques remain expensive and require qualified skilled personnel, alternatives strategies are emerging and novel technologies based on biochemical methods. These techniques are based on the interaction target-recognition element. We use in this work, a new class of biomolecules called aptamers. They are single or double stranded DNA or RNA, selected by their ability to recognise a target, by a combinatorial method of selection in vitro called SELEX (Systematic Evolution of Ligands by EXponantial enrichment).The aim of this work is the development of new methods based on aptamers for OTA determination in foodstuffs. We will develop two methods, one method for extraction and another one for detection. The first one consists in an aptamer-based affinity column or oligosorbent. This device of extraction was evaluated in term of retention, specificity, selectivity and regeneration. While the second is based on an aptasensor developed in a fully automated system. All the optimizations and manipulations were performed using automated sequences. After optimizations, these two methods are applied for OTA determination in real beer samples. The obtained limits of detection are much lower than the maximum tolerated levels of OTA in set by the European commission. Also, the high recovery yields obtained show the good applicability of the developed oligosorbent and aptasensor in real sample without matrices effect.