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

在与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检测和量化感染原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，这些技术有可能改变分子诊断（MD）的执行方式，将申请人以前的研究转化为全动态范围量化的新平台。在该提案中，两位申请人将汇集他们自己长期以来在诊断技术（Murray）和最先进的微流体技术（Castell）的学术研究和翻译方面的经验，并将与公司在分子诊断的商业开发方面的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）目标RNA/DNA的数量，以使检测成为可能。这需要热循环和监测荧光变化，这在很大程度上限制了这种设备的实验室设置与熟练的操作人员。由于供应链和设备短缺，目前的大流行也凸显了对替代医学博士的需求。另一种方法是在恒温下扩增DNA，即所谓的等温扩增。环介导扩增（LAMP）是快速的，使用4到6个引物，具有高特异性，对样品中的靶分子非常敏感，并且对污染物也相对免疫。在LAMP中检测DNA扩增最简单的方法是通过发射光来实现，这一过程被称为实时生物发光测定（BART）。该方法利用萤火虫荧光素酶将DNA扩增的副产物转化为具有光强峰值的连续光信号，其时间与原始目标浓度直接相关。BART是由申请人和ERBA Molecular公司现任CEO共同发明的。它被授权给跨国公司3M用于食品病原体检测，并提供美国农业部食品微生物学检测的首选方法。利用微流体技术在油中产生纳米级水基液滴，每个液滴形成一个用于诊断测试的反应室，有望实现小体积、快速MD。我们已经成功地展示和发表了稳定的微滴进行LAMP-BART反应，无论是独立的还是在人工细胞结构内。该项目旨在开发一个平台，通过在单次同时测试中对微滴内广泛的DNA浓度进行定量，提供准确的病原体负荷定量。由于微流体使反应液滴的数量增加，在低数量的目标DNA分子下定量的准确性增加。通过确定病原体序列提供了重要的附加诊断信息，特别是当序列变异用于跟踪疾病传播时。最近开发的拇指大小的设备（牛津纳米孔技术MinION）利用纳米孔技术使长序列能够被读取。最近有研究表明，LAMP的扩增DNA可以用该装置测序，我们已经证明了一种新的方法，可以对每个序列进行索引，该方法可以同时分析多个样本，并使特定的突变和缺失得以识别。该项目将整合这些方法，开发一个基于微流体的诊断平台，提供准确的全动态范围定量，以更低的成本获得序列信息。这可以为传染病监测和分子诊断提供显著的好处。

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.