Quantification and Viability of "Indicator" E. coli by Lab on a Chip Isothermal Nucleic Acid Amplification for Biosecurity in Sustainable Aquaculture

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Filter feeding bivalve shellfish, which include mussels, oysters, clams, and cockles, naturally accumulate microorganisms from their environment. These can pose a risk to human health after consumption, including infection with enteric viruses (e.g. Norovirus) and harmful bacteria (e.g. Salmonella Spp. and Escherichia coli), and exposure to bio-toxins produced by marine algae. Therefore, in the UK, bivalve shellfish are routinely tested in accordance with European Union regulations to ensure that they are safe to eat, and which (if any) treatment they should undergo before market. One species, Escherichia coli, is a human pathogen (disease causing microorganism) and a causative agent in gastroenteritis (food poisoning). It is also found naturally in human and animal intestines and is a valuable indicator of the level of faecal contamination in water, and therefore an indirect measure of the threat posed by other pathogens associated with human waste and sewage. The UK statutory shellfish monitoring programme is based at the Centre for Environment, Fisheries and Aquaculture Sciences' (Cefas) laboratory in Weymouth. Here, E. coli in bivalve shellfish flesh is measured using a regulatory standard technique wherein the bacteria are cultivated in the laboratory before their numbers are estimated. This method can take up to several days, allowing time for the bacteria to grow and replicate to concentrations at which they can be counted and analysed. In contrast, nucleic acid amplification is a method in which a genetic sequence from the target organism is amplified to a point at which it can be detected, analysed and counted in just a few hours and can be adapted to provide a wealth of other information about the organism (e.g. how dangerous it is). One problem with nucleic acid amplification using DNA (genomic) sequences is that these can be present in dead cells as well as those that remain active and still pose a risk to human health. This is circumvented by the use of chemical agents that destroy the DNA from dead cells before the amplification stage. Additionally, it is possible to measure whether the DNA sequence comes from a genome that is in-tact (i.e. the cell is still alive) or measure a different, but very similar molecule, mRNA, which is quickly degraded from dead cells. Most nucleic acid methods require laboratories containing bulky equipment and skilled personnel. In contrast, the Lab on a Chip concept is the miniaturisation of laboratory processes to a point at which they can be easily automated, and carried out in portable (e.g. handheld) or deployed (e.g. in the ocean) devices providing in situ and real-time analysis. Our objective is to combine nucleic acid methods for the detection of E. coli with state of the art Lab on a Chip technology to (1) provide an automated assay (test) for the measurement of E. coli from shellfish flesh in the laboratory, and (2) to prime the development of a deployable E. coli sensor that will carry out analysis of seawater in proxy to shellfish harvesting areas. This will be beneficial for two reasons. First, the new assays will aim to improve the speed and accuracy of detection. This is crucial as underestimation of the microbiological contamination in shellfish leads to increased risk for the consumer, whereas over estimation can lead to intervention and significant cost to the industry. Second, the development of a deployable E. coli sensor will enable scientists to study the routes of contamination and the environmental / seasonal events that underpin them. Whilst it is not anticipated that this system will be delivered during the lifetime of this project, the development of the Lab on a Chip nucleic acid amplification method will represent a significant step towards its completion. The technology that the proposed research will develop can be easily modified for a wealth of other applications in food safety, medical diagnostics and environmental microbiology.