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.

过滤喂食双壳类贝类（包括贻贝，牡蛎，蛤and和cock虫）自然会从其环境中积累微生物。这些可以在消费后对人类健康构成风险，包括感染肠道病毒（例如诺如病毒）和有害细菌（例如沙门氏菌属和大肠杆菌），以及暴露于海洋藻类产生的生物毒素。因此，在英国，双壳类贝类经常按照欧盟法规进行测试，以确保它们安全食用，以及（如果有）应在市场前接受的治疗。一种物种是大肠杆菌，是一种人类病原体（引起微生物的疾病）和一种胃肠炎（食物中毒）的病因。它也自然地在人类和动物肠道中发现，是水中粪便污染水平的宝贵指标，因此是对与人类废物和污水相关的其他病原体构成的威胁的间接度量。英国法定贝类监测计划基于环境，渔业和水产养殖科学中心（CEFAS）的实验室。在这里，使用调节标准技术测量双壳类贝类肉中的大肠杆菌，其中细菌在实验室估计之前在实验室中培养。这种方法最多可能需要几天，从而使细菌的成长并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，其中将来自靶基体的遗传序列放大到可以在短短几个小时内检测，分析和计数的点，并且可以适应以提供有关生物体的其他信息（例如，它的危险程度）。使用DNA（基因组）序列扩增核酸扩增的一个问题是，这些序列可以存在于死细胞中，以及那些保持活跃的细胞中，并且仍然对人类健康构成风险。通过使用化学剂在扩增阶段破坏死细胞中的DNA的化学剂可以避免这种情况。此外，可以测量DNA序列是来自室内（即细胞还活着）还是测量不同但非常相似的分子mRNA的基因组，该基因组很快从死细胞中降解。大多数核酸方法都需要装有笨重的设备和熟练人员的实验室。相比之下，芯片概念的实验室是将实验室过程的小型化，以至于它们可以轻松自动化，并在便携式（例如手持式）或部署（例如在海洋中）的设备（例如，在海洋中）进行，从而提供了原位和实时分析。 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.这将是有益的，有两个原因。首先，新测定将旨在提高检测的速度和准确性。这至关重要，因为贝类中微生物污染的低估会导致消费者的风险增加，而估算可能会导致干预和对行业的巨大成本。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和支撑它们的环境 /季节性事件。虽然没有预料到该系统将在该项目的寿命中传递，但实验室在芯片核酸扩增方法上的开发将代表其完成的重要一步。拟议研究将开发的技术可以轻松修改，以用于食品安全，医学诊断和环境微生物学方面的许多其他应用。

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.