Recombinase Polymerase Amplification for Gambierdiscus and Fukuyoa detection: a step further in the ciguatera risk management

Ciguatera fish poisoning is one of the most relevant seafood-borne illnesses worldwide. It is caused by the ingestion of fish contaminated by ciguatoxins (CTXs). Primary producers of CTXs are dinoflagellates of the genera Gambierdiscus and Fukuyoa. This study is focused on the development of bioanalytical tools for the detection of Gambierdiscus and Fukuyoa. To achieve this objective, Recombinase Polymerase Amplification (RPA), which consists of an isothermal DNA amplification during a short period (30 minutes), was combined with an enzyme-linked oligonucleotide assay (ELONA). To evaluate the specificity of the RPA-ELONA, firstly primers for the genera Gambierdiscus/Fukuyoa were exposed to genomic DNA of different species (G. australes, G. excentricus, G. belizeanus, G. balechi and F. paulensis) and other microalgae used as controls (O. cf. ovata, P. lima and C. monotis). The same genomic DNA pools were also tested with species-specific primers for Gambierdiscus australes and Gambierdiscus excentricus. Finally, DNA was extracted from single cells of the previously mentioned genera and species, and tested with all the primer sets. For both the experiments, detection was achieved only when combining capture probes with their target RPA product, and no significant responses were observed in the presence of non-target DNA. Obtained results demonstrate the ability of the system to discriminate not only the genus Gambierdiscus/Fukuyoa from other microalgae, but also G. australes and G. excentricus species from their congeners. Furthermore, the limit of detection is as low as a single cell. Acknowledgments: The research has received funding from the Ministerio de Ciencia, Innovacion y Universidades through the CIGUASENSING (BIO2017-87946-C2-2-R) project. The authors acknowledge support from CERCA Programme/Generalitat de Catalunya. G. Gaiani acknowledges IRTA-Universitat Rovira i Virgili-Banco Santander for her PhD grant (2018PMF-PIPF-19).