'Point of Sample' Genetic Sensor Instrumentation to Improve Biosecurity In UK Shellfisheries and Bathing Waters

This project is focused on creating new methods and technology that will reduce the risk of human disease contracted through eating shellfish, or from contact with contaminated water supplies. To do this, it will develop new tools to enable regulatory / governmental authorities (such as the Food Standards Agency, Centre for Environment, Fisheries and Aquaculture Science, and the Environment Agency) to rapidly and accurately measure pathogens (such as viruses, bacteria, and toxin-producing algae) in aquatic environments, and the food cultivated within them. These new tools (together with conventional methods) will enable these end-users to examine where pathogens in the environment and shellfish come from, how they move and how they are modified by environmental conditions. The tools that the project will develop use nucleic acid amplification to measure pathogens. Nucleic acids contain the genetic information specific to each species and are used in their replication and function. Therefore, measuring how much of a specific sequence is present gives pathogen identification, quantification and can tell us about their activity (e.g. are they producing a toxin). It is now possible to amplify (i.e. copy) even just a single molecule of a target nucleic acid, increasing its concentration to a point when it can be detected and quantified. However, there are problems. Specifically: (1) even within a single species there are often significant differences between the nucleic acid sequences present in individuals; (2) one form of nucleic acid, called DNA, can persist after death and hence measuring DNA alone often leads to over estimation of the number of living individuals; and (3) the accuracy of the measurement is often sensitive to how the sample is prepared (e.g. different days, equipment and people). This project will overcome these problems by: (1) using Next Generation Sequencing methods to realise the variability of nucleic acid sequences in a single species to ensure that our amplification methods can cope with this variability; (2) looking at short-lived nucleic acids called RNA, as well as DNA, to see if the pathogen is alive and active; and (3)automating the process of preparing samples for measurements to improve accuracy . The project will develop what is known as Microfluidic Lab on a Chip (MFLOC) technologies. These reduce the scale of analytical processes traditionally performed in the lab. For example, miniature pipes (typically one tenth of a millimetre across) together with miniature pumps, valves and optics are used to take in sample, mix it with a chemical, and to measure the resultant changes (e.g. a change in colour or fluorescence). In this project, MFLOC will be used to measure nucleic acids in water samples taken from shellfisheries and other natural water sources, and in shellfish flesh samples. It will do this by mixing a small volume of sample containing purified nucleic acids with dehydrated reaction mixtures containing all of the necessary reagents to support amplification and detection of specific nucleic acid sequences, which are unique to the target species. This occurs within a micro-chamber on a disposable cartridge or 'chip', which fits into a specialist device which controls and measures the amplification reaction in real time. The use of this technology makes this kind of complex and state of the art analysis easy to automate, with corresponding increases in efficiency and reproducibility. This technology is easily adapted for the measurement of potentially any species providing that at least some genetic sequence information is known, and therefore has many potential applications beyond the scope of this specific project.