Paper-based platform for on site, rapid, and multiplexed DNA-based pathogen detection in aquaculture

Shellfish has the potential to become one of the largest future mainstream food in the UK due to its health, ethical and sustainable benefits. Oysters, mussels and clams are recognised as environmentally friendly and sustainable compared to other types of farming. In the UK, the shellfish sector was worth £11.7 million in 2016 with a signification growth of 60% in the past 10 years, reaching its highest level of 7,732 tonnes in 2016. Shellfish is also a sustainable food source. They do not require feeding, as they filter nutrients from the water around them. More importantly, contrary to fish, they do not require space or enrichment in order to grow, and their farmed habitat is similar to their wild habitat. They also often do not require much cooking, e.g. oysters are often eaten raw. However, this latter advantage leads to a historical issue in the shellfish industry: the product has a very short lifecyle.

To ensure the product is safe for consumption, food safety programmes perform extensive testing on shellfish to detect human pathogens that could have accumulated from contamination of the water (since shellfish feed by filtering the water, pathogens such as Salmonella or norovirus can accumulate even if they are at low concentration in the water). The conventional testing procedures however are time-consuming and laborious, and require the access to sophisticated laboratory facilities. When the results of the test are finally obtained, the shellfish has been already consumed, which limits the implementation of any preventive action. This resulted in nearly 1 million cases of food poisoning in the UK in 2011, leading to about 11 million lost working days.

In this project, we will develop and implement a low-cost technique, which has been developed for the detection of human and animal pathogens in low-resource settings, such as in the field in Uganda and in a farm in India, for the rapid detection of pathogens in seawater in aquaculture. Together with our partners the Centre for Environment, Fisheries and Aquaculture Sciences Cefas (the world leader in marine science and technology) and Cromarty Mussels Ltd (the largest single site for both mussel and oyster farming in Scotland), we will develop deployable paper-based biosensor devices (lab-on-a-paper) for the online monitoring of seawater and the detection of foodborne pathogens.
The platform relies on the enrichment of pathogens from seawater using a combination of filtration and magnetic beads in a syringe, onto a paper-based biosensor. By folding the paper, in a process akin to origami, the genetic material of the pathogens is purified and distributed into specific areas, where nucleic acids are amplified. This amplification (performed using a small hand-held heater with low power at around 60C, but could also be performed in a thermos) is then detected using either direct visualisation of a color change or using a mobile phone for quantification, within 1h.

Used in farms, the platform will allow the rapid detection of 3 pathogens, Salmonella and two types of norovirus, which are the most common pathogens associated with illness from shellfish consumption. Together with our partners, we will test the performance of the devices in the field in a shellfish farm, in Inverness. These results will then enable the consortium to progress towards developing this as a product for the industry to be able to rapidly respond to contamination events, so that consumer safety is assured, and shellfish aquaculture can grow to its full potential. Deployed within the environment, this platform also has the potential to serve as an early warning system of contamination events and to enable source disease tracking and thus risk prediction.

Bivalve molluscan shellfish concentrate contaminants from the water in which they grow, which may cause human illness upon consumption. The risk is increased by the fact that some shellfish are eaten raw (oysters) or lightly cooked (mussels). This resulted in nearly 1m cases in the UK in 2011, leading to about 11m lost working days.
To limit the risk, food safety agencies conduct regular testing of farms and shellfish production, by harvesting shellfish and sending them to specialist laboratories, where the test is performed. Current tests rely on the use of E.Coli as a proxy marker for human pathogen contaminations and their numbers are detected through culture-based assays. The whole process can take days, such that when the results are received at the farm, the product has already been consumed. New technologies such as real-time PCR can provide results more rapidly but cannot be performed on site, resulting in similar delays.

This project thus aims at answering two key challenges in aquaculture:
- The need for portable technologies to test the quality of seawater and shellfish, which typically are located in remote areas with little access to sophisticated labs.
- The necessity of shortening the duration of the procedures due to the short life of these products and the high risk associated with consumption of contaminated product.

In this project, we will translate low-cost, low-power and point-of-use paper-based biosensors, which have already been developed for human healthcare and veterinary diagnostic applications in low-resource settings, to monitor food safety in aquaculture. Together with our partners the Centre for Environment, Fisheries and Aquaculture Sciences Cefas (world leader in marine science and technology) and Cromarty Mussels Ltd (the largest single site for both mussel and oyster farming in Scotland), we will validate their application for the online monitoring and rapid detection of foodborne pathogens at the farm.

The project aims to create impact within society by enabling the rapid detection and identification of human pathogens within shellfish aquaculture. This will have a direct impact on the safety of the food produced, thus limiting the health consequence that can result from the consumption of contaminated food (a significant issue for the UK, causing millions of days off work each year). This issue arises due to the long process required for testing shellfish in aquaculture, which can take days and is often performed offsite. Due to their short product life cycle, when the results of the tests are known, the product has already been consumed. Public health consequences also lead to economic issues, as food quality is a primary concern of the sector and a change in quality can severely impact farmers, who then have difficulties in selling their product. A mitigating solution exists through processes such as relaying or depuration, which make use of the natural filtering capabilities of shellfish to decrease the potential contamination. But these processes can take days and require additional infrastructure, both factors leading to significant costs. Depuration is also not efficient against viruses.

The platform developed during this project to rapidly detect pathogens in seawater in aquaculture promises to deliver significant impact in enabling direct testing at the site. As a portable field test, we will also explore its application to monitor the effectiveness of the depuration process of shellfish. Deployed to the environment, this platform also has the potential to serve as an early warning system of contamination events and to enable source disease tracking and thus risk prediction.

The project will be conducted in close collaboration with two non-academic partners: the Centre for Environment, Fisheries and Aquaculture Sciences, Cefas (world leader in marine science and technology) and Cromarty Mussels Ltd (the largest single site for both mussel and oyster farming in Scotland). Their input in the project will ensure that the technologies developed will be efficiently translated into practical use during this project. Cefas will work with regulators (e.g. FSA) to ensure that the project develops in line with regulations, but also to explore how it could influence new regulations to enable the benefits of rapid on-site testing. In practice, this impact will be realised through the development of commercial devices and intellectual property, the delivery of which will have additional economic impacts.

Academics and commercial organisations will benefit in the short term. Academics involved in the immediate environment of the Investigators will benefit from the improved capabilities of this technology and its demonstration including: Professor R. Zadocks (Moredun Institute and Life Sciences Glasgow); Professor Elise Cartmell (Scottish Water), and Professor Marian Scott (Maths and Stats - Glasgow).

This research is highly multi-disciplinary at the intersection of molecular biology, aquaculture, microfluidics, and biosensing. A broad range of other academics will benefit, including those involved in sensor instrumentation, microfluidics and microsystems, and environmental monitoring, as the new capabilities of rapid pathogen detection are delivered. Commercial organisations, including fisheries and testing laboratories as our partners will also benefit in the short term, through the availability of new tools for rapid testing.