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

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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.