Resumen de Development of bioanalytical devices for the detection of ciguatoxins and the ciguatoxin producing genera gambierdiscus and fukuyoa
Ciguatera fish poisoning (CFP) is a foodborne disease that can cause gastrointestinal, cardiological and neurological symptoms that can last weeks, months or even years and in some cases leads to death. It is caused by the ingestion of fish containing ciguatoxins (CTXs), a group of cyclic polyether lipophilic compounds produced by microalgae of the genera Gambierdiscus and Fukuyoa, which accumulate into fish flesh and through the food webs. Several fish species are implicated in CFP, and discriminating between contaminated and uncontaminated specimens is an important challenge, since toxic specimens do not look, smell or taste any differently from non-toxic ones. Thus, taking into consideration that an antidote for CFP has not been found yet, the efforts of the scientific community must focus on the prevention, by providing fast and reliable tools for the detection not only of CTXs in fish and algal samples, but also of the Gambierdiscus and Fukuyoa CTX producing species in the environment.
The main goal of this doctoral thesis is to provide biotechnological tools for the characterization of the risk of CFP in order to promote food safety and human health. Particularly, the major focus is the development of bioanalytical devices for the detection of different Gambierdiscus and Fukuyoa species and of CTXs. Additionally, this thesis aims at providing fast and reliable strategies to shorten and simplify the sample pretreatment necessary for the analysis of environmental samples. In general terms, this thesis intends to demonstrate the applicability of reliable biotechnological tools, which can be easily implemented in portable devices, paving the way for the in situ detection that would speed up monitoring analysis.
In order to achieve this objective, this thesis reports the development of fast extraction techniques for DNA and CTXs with the use of portable devices. Then, on one hand, it shows how primers modified with tails were exploited to perform both recombinase polymerase amplification (RPA) and PCR to simultaneously amplify DNA from the genera Gambierdiscus and Fukuyoa and from more than one toxin producing species. On the other, the application of antibodies that target four main CTXs belonging to two groups of congeners (CTX1B and CTX3C) is described for their combined or separated detection. At first, the systems were characterized with colorimetric assays, and then they were integrated in the development of electrochemical biosensors for the detection of DNA belonging to two Gambierdiscus species at the time or of CTXs. Finally, this thesis provides a description of how the developed systems were applied to the analysis of natural samples, comparing the results to well-established techniques.
