Resumen de Dna-based assays and biosensors for the detection of toxic microalgae and viruses in the marine environment
The presence of biological hazards such as toxic microalgae and viruses in the marine environment has become a serious concern due to their direct impact on marine life, human health and economic-related activities. Nonetheless, the application of monitoring programs can help to prevent and mitigate their impacts. Fostered by the need to have alternative methods to current light microscopy and conventional molecular methods, the development of rapid, reliable, user-friendly and in-situ analysis tools is of current interest in environmental research.
The general objective of this thesis is the development of DNA-based assays and biosensors for the detection of marine toxic microalgae and viruses, and their application to the analysis of environmental samples. Moreover, this thesis aims to contribute to other steps in the environmental analysis process, including sample pre-treatment and DNA extraction.
To achieve this goal, the following specific objectives have been defined: • To establish a rapid and simple DNA extraction method for microalgae.
• To develop qPCR assays to detect microalgae.
• To exploit isothermal recombinase polymerase amplification and sandwich hybridisation formats to develop colorimetric assays and electrochemical biosensors for microalgae and viruses.
• To evaluate the applicability of the DNA-based assays and biosensors to the analysis of environmental (planktonic, benthic and spiked) and oyster samples.
• To develop a magnetic bead (MB)-based strategy to capture and concentrate viral particles.
This thesis has the following structure: • Chapter 1 contains a general introduction, which includes a brief description of the toxic marine microalgae and viruses. It describes how relevant the development of new methods is and provides the state of the art of DNA-based tools for their detection, focusing on those based on isothermal amplification techniques. Moreover, an overview of the key factors in the development and implementation of DNA-based tools is provided.
• Chapter 2 includes the general and specific objectives of this thesis. A list of the scientific publications achieved through this thesis, together with the personal contribution, is provided.
• Regarding the experimental part, this thesis has been divided into three different sections according to the target analyte: • Chapter 3 is focused on the development of qPCR and colorimetric assays to detect the microalgae Karlodinium. Chapter 3A describes the development of a qPCR assay to detect and discriminate between K. veneficum and K. armiger, and its subsequent application to environmental planktonic samples. The establishment of a new DNA extortion method is also addressed. In Chapter 3B, the development of a colorimetric assay exploiting RPA to detect and discriminate between K. veneficum and K. armiger is pursued. The use of different approaches (synthetic DNA, genomic DNA or cells) to construct the calibration curves is explored.
• Chapter 4 describes the development of colorimetric assays and electrochemical biosensors, both exploiting RPA, to detect the microalgae Ostreopsis. Chapter 4A is focused on the development of a colorimetric assay to detect and discriminate between O. cf. ovata and O. cf. siamensis. Primer design for RPA and the removal of the cleaning step after RPA is examined. Moreover, a predictive model is proposed to evaluate the relationship between this system and microscopy counts in the analysis of environmental planktonic and benthic samples. In Chapter 4B, the development and application of a MB-based electrochemical biosensor for O. cf. ovata is described.
• Chapter 5 is focused on the development of electrochemical biosensors and MB-based capture strategies for ostreid herpesvirus-1. Chapter 5A describes the development of an electrochemical biosensor exploiting RPA and its application to the analysis of oysters. In this case, gold electrodes are used as DNA immobilisation supports. In Chapter 5B, the use of MBs able to capture and concentrate viable viral particles from seawater and oyster tissue homogenate is presented. Experimental oyster infections as well as DNA and RNA analysis by qPCR are covered.
• In Chapter 6, a general discussion of the findings achieved in this thesis is provided.
• Finally, Chapter 7 summarises the general conclusions of the thesis and the future perspectives and applications of this research.
The findings achieved in this thesis lead to draw the following conclusions: • Different tools based on DNA including qPCR, colorimetric assays, electrochemical biosensors and MB-based capture strategies have been developed to detect Karlodinium, Ostreopsis and ostreid herpesvirus-1 (OsHV-1), and successfully applied to the analysis of environmental samples (seawater and oyster samples).
• A rapid and simple method to extract DNA has been applied to different microalgae species, and successfully coupled with qPCR for the quantitative analysis of K. veneficum and K. armiger.
• The use of isothermal recombinase polymerase amplification (RPA) with tailed primers, followed by a sandwich hybridisation assay, has been proved useful to develop both colorimetric assays and electrochemical biosensors.
• Primer design has been demonstrated to be a key factor for RPA performance. Furthermore, the optimisation of primer concentration in the RPA enabled to avoid a purification step of amplified products before their detection. • The design of species-specific primer sets for two Karlodinium and two Ostreopsis species enabled to develop dual qPCR and colorimetric systems to detect, quantify and discriminate between K. veneficum and K. armiger as well as O. cf. ovata and O. cf. siamensis.
• The use of MBs as immobilisation supports resulted useful for the development of the electrochemical biosensor for O. cf. ovata, offering advantages in terms of improved washing steps, ease of handling and regeneration of electrodes.
• The developed methods showed enough specificity to be applied in surveillance activities. Additionally, qPCR, colorimetric assays and electrochemical biosensors developed for Karlodinium and Ostreopsis achieved LODs below the prosed current thresholds.
• The use of genomic DNA demonstrated to perform better than synthetic DNA. In particular, both the use of genomic DNA coming from a pool of cells or from cell dilutions proved useful to construct calibration curves.
• DNA quantifications provided by the developed molecular tools and qPCR were in good agreement. A high degree of correlation between cell abundances provided by the developed molecular methods and microscopy was obtained. In addition, the construction of a predictive regression model allowed to estimate cell abundances from DNA quantifications.
• MBs coated with an anionic polymer have been successfully used to capture viable OsHV-1 form both seawater and oyster tissue homogenate samples, and to concentrate OsHV-1 from the homogenate.
Future work: • To apply the DNA-based methods to a higher number of samples, also coming from different geographical areas, to further validate these new approaches.
• To develop multiplex systems (one-spot reaction) for the dual qPCR and colorimetric assays by mixing the primers in the same amplification reaction and using different labels/physical separation for the sequent detection.
• To decrease the LODs of the electrochemical biosensor for OsHV-1 by improving the amplification and the detection steps and/or using pre-concentrating agents such as MBs.
• To use MBs in combination with qPCR to capture and detect OsHV-1 in seawater samples from aquaculture facilities.
Potential applications: • The use of MBs to obtain purified and viable OsHV-1 for research activities.
• The implementation of the developed bioanalytical tools, after proper validation and inter-laboratory studies, in environmental monitoring activities.
• The integration of the developed methods into microfluidics platforms and easy-to-handle compact devices for their deployment into the market.
Overall, this thesis demonstrates the opportunities that nucleic acids offer to environmental analytical chemistry by providing new approaches that will pay the way towards the implementation of rapid, specific, simple, low-cost and in situ analysis tools to improve research, monitoring and management of marine biological hazards.
