Platform technology for full dynamic range infectious disease detection and quantification.

用于全动态范围传染病检测和量化的平台技术。

• 批准号: BB/W00335X/1
• 负责人: James Murray
• 金额: $ 25.47万
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW00335X%2F1
• 关键词: Platform; technology; full; dynamic; range

In the fight against infectious diseases such as COVID-19 and tuberculosis, molecular diagnostics is the essential tool for detecting and quantifying infectious agents through their DNA or RNA, hence diagnosing disease.This proposal will develop and integrate several innovative technologies that together have the potential to transform the way in which molecular diagnostics (MD) is performed, translating the applicants' previous research into a novel platform for full dynamic range quantification. In this proposal, the two applicants bring together their own longstanding experience of academic research and translation of diagnostics technologies (Murray) and state-of-the-art microfluidics (Castell), and will link with company experts in the commercial development of molecular diagnostics and public health infectious disease experts. Whilst the approaches are equally applicable to most infectious diseases, we will focus on variants of SARS-CoV-2. The most common method in MD is the polymerase chain reaction (PCR), using repeated temperature cycling and a pair of short specific DNA primers to increase exponentially (amplify) the amount of the targeted RNA/DNA to enable detection. This requires thermal cycling and monitoring of fluorescence changes, which largely limits such devices to laboratory settings with skilled operators. The current pandemic has also highlighted the need for alternative MDs due to supply chain and equipment shortages. Another approach is to amplify DNA at a constant temperature, so called isothermal amplification. Loop-mediated amplification (LAMP) is rapid, uses 4 to 6 primers giving high specificity, and is very sensitive to target molecules in a sample, and is also relatively immune to contaminants.Detecting DNA amplification in LAMP is most simply achieved through the emission of light in a process known as the bioluminescent assay in real-time (BART). This uses firefly luciferase to convert a by-product of DNA amplification into a continuous light signal with a peak in light intensity whose timing is directly related to the original target concentration. BART was co-invented by the applicant and the now CEO of ERBA Molecular. It is licensed to multinational 3M for food pathogen detection and provides the preferred method of food microbiology testing of the US Department of Agriculture. The promise of low volume, rapid MD may be achievable using microfluidics to generate nanolitre water-based droplets in oil, each forming a reaction chamber for a diagnostic test. We have successfully demonstrated and published stable micro-droplets carrying out LAMP-BART reactions both independently and inside artificial cell structures. The project seeks to develop a platform to provide accurate quantification of pathogen load through the quantification of a wide range of DNA concentrations within micro-droplets in a single simultaneous test. The accuracy of the quantification at low numbers of target DNA molecules is increased due to the number of reaction droplets enabled by microfluidics.Important additional diagnostic information is provided through determining pathogen sequences, particularly when sequence variants are used to track the disease spread. The recently developed thumb-sized device (Oxford Nanopore Technologies MinION) utilises nanopore technology to enable long sequences to be read. Recently it has been shown that amplified DNA from LAMP can be sequenced with this device, and we have demonstrated a new method of indexing each sequence read that can be used to simultaneously analyse multiple samples and enable specific mutations and deletions to be identified.This project will integrate these approaches and develop a microfluidic-based diagnostics platform providing accurate full dynamic range quantification linked to the ability to obtain sequence information at lower cost. This can offer significant benefits for infectious disease monitoring and molecular diagnostics.

在抗击COVID-19和结核病等传染病的斗争中，分子诊断是通过其DNA或RNA检测和定量感染因子从而诊断疾病的重要工具。该提案将开发和整合几种创新技术，这些技术共同具有改变分子诊断（MD）执行方式的潜力，将申请人先前的研究转化为用于全动态范围量化的新颖平台。在这项提案中，两位申请人将自己在诊断技术（Murray）和最先进的微流体技术（Castell）的学术研究和翻译方面的长期经验结合在一起，并将与公司分子诊断商业开发专家和公共卫生传染病专家建立联系。虽然这些方法同样适用于大多数传染病，但我们将重点关注SARS-CoV-2的变体。MD中最常见的方法是聚合酶链反应（PCR），使用重复的温度循环和一对短的特异性DNA引物以指数方式增加（扩增）靶向RNA/DNA的量以进行检测。这需要热循环和荧光变化的监测，这在很大程度上限制了这种设备与熟练的操作员的实验室设置。由于供应链及设备短缺，目前的疫情亦突显出对替代医疗器械的需求。另一种方法是在恒定温度下扩增DNA，即所谓的等温扩增。环介导扩增（LAMP）是快速的，使用4至6个引物，提供高特异性，并且对样品中的靶分子非常敏感，并且对污染物也相对免疫。这使用萤火虫荧光素酶将DNA扩增的副产物转化为具有光强度峰值的连续光信号，其定时与原始靶浓度直接相关。BART由申请人和ERBA分子公司现任首席执行官共同发明。它被授权给跨国公司3 M进行食品病原体检测，并为美国农业部提供食品微生物检测的首选方法。使用微流体技术在油中产生纳升水基液滴，每个液滴形成用于诊断测试的反应室，可以实现低体积、快速MD的前景。我们已经成功地证明并发表了独立地和在人工细胞结构内进行LAMP-BART反应的稳定微滴。该项目旨在开发一个平台，通过在一个单一的同时测试中定量微滴内的各种DNA浓度来提供病原体负荷的准确定量。由于微流控技术能够实现反应液滴的数量，因此在低数量靶DNA分子下的定量准确性得以提高。通过确定病原体序列提供了重要的额外诊断信息，特别是当序列变体用于跟踪疾病传播时。最近开发的拇指大小的设备（Oxford Nanopore Technologies MinION）利用纳米孔技术来读取长序列。最近已经表明，可以用该装置对来自LAMP的扩增DNA进行测序，我们已经展示了一种新的索引每个序列读段的方法，该方法可用于同时分析多个样品，并能够识别特定的突变和缺失。该项目将整合这些方法，并开发一种微流体-基于的诊断平台，提供与以较低成本获得序列信息的能力相关的准确的全动态范围量化。这可以为传染病监测和分子诊断提供显著的益处。

项目成果

Plant Genotyping - Methods and Protocols

植物基因分型 - 方法和方案

• DOI: 10.1007/978-1-0716-3024-2_20
• 发表时间: 2023
• 作者: Hardinge P

Optimized Loop-Mediated Isothermal Amplification (LAMP) Allows Single Copy Detection Using Bioluminescent Assay in Real Time (BART).

优化的环介导等温扩增 (LAMP) 允许使用实时生物发光测定 (BART) 进行单拷贝检测。

• DOI: 10.1007/978-1-0716-2453-1_8
• 发表时间: 2022
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Hardinge P

VarLOCK - sequencing independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance

VarLOCK - 独立于测序、快速检测关注点检测、qPCR 管道和国家废水监测的 SARS-CoV-2 变体

• DOI: 10.1101/2022.01.06.21268555
• 发表时间: 2022
• 作者: Nan X