DNA-based assays and biosensors for the detection of toxic microalgae and viruses in the marine environment

Identifier:  TDX:3008

Handle: https://hdl.handle.net/20.500.11797/TDX3008

Authors:  Toldrà Filella, Anna

Abstract:
This thesis aims to contribute to the development and applicability of DNA-based assays and biosensors for the detection of toxic marine microalgae (i.e. Karlodinium and Ostreopsis) and viruses (i.e. ostreid herpesvirus-1, OsHV-1), as well as to other steps in the environmental analysis process, such as sample pre-treatment and DNA extraction. These new DNA-based approaches include qPCR assays, colorimetric assays, electrochemical biosensors and MB-based capture strategies. A qPCR assay for the detection of Karlodinium species, combined with a simple and rapid DNA extraction method, is described. The variety of colorimetric assays and electrochemical biosensors developed for the detection of species of Karlodinium, Ostreopsis and/or OsHV-1 exploit the isothermal recombinase polymerase amplification technique, tailed primers and a sandwich hybridisation detection format. Primer design for microalgae and viruses and optimisation of RPA and qPCR parameters are covered. Magnetic beads are exploited as immobilisation supports in the development of electrochemical biosensors, and also as an OsHV-1 capture and pre-concentration strategy. The applicability of the developed DNA-based tools to the analysis of microalgae-contaminated seawater/macroalgae environmental samples and OsHV-1 contaminated seawater/oyster samples is demonstrated by their comparison with well-established techniques (i.e. light microscopy and/or qPCR). The vision underlying this work is to contribute to pave the way towards the implementation of rapid, specific, easy-to-use, low-cost and in situ analysis tools to eventually improve research, monitoring and management of marine biological hazards.