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

通过芯片实验室等温核酸扩增对“指标”大肠杆菌进行定量和活力，以实现可持续水产养殖的生物安全

• 批准号: BB/M025837/1
• 负责人: Matt Mowlem
• 金额: $ 31.34万
• 依托单位: NATIONAL OCEANOGRAPHY CENTRE
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM025837%2F1
• 关键词: Quantification; Viability; Indicator; E.; coli

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.

滤食性双壳贝类，包括贻贝、牡蛎、蛤和鸟蛤，自然地从它们的环境中积累微生物。这些食品在食用后会对人体健康构成风险，包括感染肠道病毒（如诺如病毒）和有害细菌（如沙门氏菌）。和大肠杆菌），以及接触海藻产生的生物毒素。因此，在英国，双壳贝类根据欧盟法规进行常规测试，以确保它们可以安全食用，以及它们在上市前应进行的处理（如果有的话）。其中一个物种，大肠杆菌，是人类病原体（致病微生物）和胃肠炎（食物中毒）的病原体。它也存在于人类和动物的肠道中，是水中粪便污染程度的重要指标，因此是衡量与人类废物和污水有关的其他病原体所构成威胁的间接指标。英国法定贝类监测计划设在韦茅斯的环境、渔业和水产科学中心（Cefas）实验室。给你E双壳贝类肉中的大肠杆菌是使用监管标准技术测量的，其中细菌在估计其数量之前在实验室中培养。这种方法可能需要几天的时间，使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，其中来自靶生物体的基因序列被扩增到可以在短短几个小时内检测，分析和计数的点，并且可以适应于提供关于生物体的大量其他信息（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细胞以及那些保持活性并仍对人类健康构成风险的细胞中。这是通过使用化学试剂来规避的，这些化学试剂在扩增阶段之前破坏死细胞的DNA。此外，还可以测量DNA序列是否来自完整的基因组（即细胞仍然活着），或者测量不同但非常相似的分子mRNA，该分子从死细胞中迅速降解。大多数核酸方法需要包含庞大设备和熟练人员的实验室。相比之下，芯片实验室概念是将实验室过程简化到可以轻松自动化的程度，并在便携式（例如手持）或部署（例如在海洋中）设备中进行，提供现场和实时分析。本研究的目的是联合收割机检测大肠杆菌。大肠杆菌的最先进的芯片实验室技术，以（1）提供一个自动化的测定（测试），用于测量大肠杆菌。大肠杆菌，以及（2）启动开发可部署的E.大肠杆菌传感器，将进行海水分析的替代贝类捕捞区。这将是有益的，原因有二。首先，新的检测方法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，而高估可能导致干预和行业的重大成本。第二，开发了一种可部署的E。大肠杆菌传感器将使科学家能够研究污染的途径和环境/季节性事件，这些事件是污染的基础。虽然预计该系统不会在本项目的生命周期内交付，但芯片实验室核酸扩增方法的开发将代表其完成的重要一步。拟议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的其他应用。

项目成果

Corrigendum to "A highly specific Escherichia coli qPCR and its comparison with existing methods for environmental waters" [Water Res. 126, 101-110].

“高度特异性的大肠杆菌 qPCR 及其与环境水域现有方法的比较”的勘误表 [Water Res。

• DOI: 10.1016/j.watres.2019.06.070
• 发表时间: 2019
• 期刊: Water research
• 影响因子: 12.8
• 作者: Walker DI

A novel portable filtration system for sampling and concentration of microorganisms: Demonstration on marine microalgae with subsequent quantification using IC-NASBA.

一种用于微生物采样和浓缩的新型便携式过滤系统：海洋微藻的演示以及随后使用 IC-NASBA 的定量。

• DOI: 10.1016/j.hal.2018.03.006
• 发表时间: 2018
• 期刊: Harmful algae
• 影响因子: 6.6
• 作者: Loukas CM

Advancing Observation of Ocean Biogeochemistry, Biology, and Ecosystems With Cost-Effective in situ Sensing Technologies

利用具有成本效益的原位传感技术推进海洋生物地球化学、生物学和生态系统的观测

• DOI: 10.3389/fmars.2019.00519
• 发表时间: 2019
• 期刊: Frontiers in Marine Science
• 影响因子: 3.7
• 作者: Wang, Zhaohui Aleck;Moustahfid, Hassan;Mueller, Amy V.;Michel, Anna P.;Mowlem, Matthew;Glazer, Brian T.;Mooney, T. Aran;Michaels, William;McQuillan, Jonathan S.;Robidart, Julie C.
• 通讯作者: Robidart, Julie C.