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

滤食双壳类贝类，包括贻贝、牡蛎、文蛤和贝类，自然会从它们的环境中积累微生物。食用后可能对人体健康构成风险，包括感染肠道病毒(如诺沃克病毒)和有害细菌(如沙门氏菌)。和大肠杆菌)，以及暴露于海洋藻类产生的生物毒素。因此，在英国，双壳类贝类根据欧盟的法规进行例行检测，以确保它们可以安全食用，以及它们在上市前应该接受哪些处理(如果有的话)。一种是大肠杆菌，它是一种人类病原体(致病微生物)，也是胃肠炎(食物中毒)的病原体。它也自然存在于人和动物的肠道中，是水中粪便污染程度的宝贵指标，因此是与人类排泄物和污水有关的其他病原体构成的威胁的间接衡量标准。英国法定的贝类监测项目设在韦茅斯的环境、渔业和水产养殖科学中心(CEFAS)实验室。在这里，双壳类贝类鱼肉中的大肠杆菌是使用一种监管标准技术进行测量的，这种技术在估计细菌数量之前在实验室进行细菌培养。这种方法可能需要几天的时间，让细菌有时间生长和复制到可以计数和分析的浓度。相比之下，核酸扩增是一种方法，在这种方法中，来自目标生物体的基因序列被放大到可以在短短几个小时内被检测、分析和计数的点，并且可以被修改以提供关于该生物体的丰富的其他信息(例如，它有多危险)。使用DNA(基因组)序列进行核酸扩增的一个问题是，这些序列可能存在于死亡细胞中，也可能存在于那些仍然活跃但仍对人类健康构成风险的细胞中。这是通过使用化学试剂在扩增阶段之前摧毁死亡细胞的DNA来规避的。此外，还可以测量DNA序列是否来自未接触的基因组(即细胞仍然活着)，或者测量不同但非常相似的分子--信使核糖核酸，而信使核糖核酸是从死亡细胞中迅速降解的。大多数核酸方法需要拥有庞大设备和熟练人员的实验室。相比之下，芯片实验室的概念是将实验室过程微型化到可以很容易地实现自动化的程度，并在提供现场和实时分析的便携式(例如手持)或部署(例如海洋)设备中进行。我们的目标是将检测大肠杆菌的核酸方法与最先进的芯片实验室技术相结合，以(1)提供一种在实验室测量贝肉中大肠杆菌的自动化分析(测试)，以及(2)准备开发一种可部署的大肠杆菌传感器，该传感器将对贝类捕捞区的海水进行分析。这将是有益的，原因有两个。首先，新的检测方法将旨在提高检测的速度和准确性。这一点至关重要，因为低估贝类中的微生物污染会增加消费者的风险，而高估则可能导致干预和该行业的重大成本。其次，可部署的大肠杆菌传感器的开发将使科学家能够研究污染的途径以及支撑它们的环境/季节性事件。虽然预计该系统不会在该项目的生命周期内交付，但芯片上实验室核酸扩增方法的开发将代表着朝着完成该方法迈出的重要一步。拟议中的研究将开发的技术可以很容易地进行修改，以用于食品安全、医疗诊断和环境微生物学等领域的大量其他应用。

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.