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

由于其健康、道德和可持续的好处，贝类有可能成为英国未来最大的主流食品之一。与其他类型的养殖相比，牡蛎、贻贝和蛤蜊被认为是环保和可持续的。在英国，贝类行业在2016年价值1170万英镑，在过去10年里增长了60%，在2016年达到了7732吨的最高水平。贝类也是一种可持续的食物来源。它们不需要喂食，因为它们从周围的水中过滤营养物质。更重要的是，与鱼类相反，它们不需要空间或丰富的环境来生长，它们的养殖栖息地与野生栖息地相似。它们通常也不需要太多的烹饪，例如牡蛎通常是生吃的。然而，后一种优势导致了贝类行业的一个历史问题：产品的生命周期非常短。为了确保产品可安全消费，食品安全规划对贝类进行了广泛的检测，以检测可能因水污染而积累的人类病原体（由于贝类通过过滤水来喂养，沙门氏菌或诺如病毒等病原体即使在水中浓度很低也会积累）。然而，传统的检测程序既费时又费力，而且需要使用先进的实验室设施。当最终获得测试结果时，贝类已经被食用，这限制了任何预防措施的实施。这导致了2011年英国近100万例食物中毒，导致大约1100万个工作日的损失。在这个项目中，我们将开发和实施一种低成本技术，该技术已被开发用于在资源匮乏的环境中检测人类和动物病原体，例如在乌干达的实地和印度的一个农场，用于快速检测水产养殖海水中的病原体。与我们的合作伙伴环境、渔业和水产养殖科学中心Cefas（世界海洋科学和技术的领导者）和Cromarty贻贝有限公司（苏格兰最大的贻贝和牡蛎养殖基地）一起，我们将开发可部署的纸质生物传感器设备（纸上实验室），用于在线监测海水和检测食源性病原体。该平台依靠将过滤和注射器中的磁珠结合在一起，将海水中的病原体富集到纸基生物传感器上。在类似于折纸的过程中，通过折叠纸张，病原体的遗传物质被纯化并分布到特定区域，在那里核酸被扩增。这种放大（使用一个小的手持式低功率加热器在60℃左右进行，但也可以在保温瓶中进行）然后使用直接可视化颜色变化或使用移动电话进行定量检测，在1小时内。该平台用于养殖场，将允许快速检测三种病原体，沙门氏菌和两种诺如病毒，这是与食用贝类疾病相关的最常见病原体。我们将与合作伙伴一起，在因弗内斯的一个贝类养殖场实地测试这些设备的性能。这些结果将使该联盟能够将其开发为一种产品，使该行业能够迅速应对污染事件，从而确保消费者的安全，并使贝类水产养殖能够充分发挥其潜力。该平台部署在环境中，也有可能作为污染事件的早期预警系统，并实现源疾病跟踪，从而实现风险预测。

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.