Novel strategies for single step molecular diagnostics assays with full dynamic range quantitation

具有全动态范围定量的单步分子诊断测定的新策略

• 批准号: BB/L022346/1
• 负责人: James Murray
• 金额: $ 50.11万
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FL022346%2F1
• 关键词: Novel; strategies; single; step; molecular

Molecular diagnostics is the most sensitive technique to detect specific organisms, pathogens or other biological material that contains DNA, and is widely used in diagnostic laboratories worldwide. It relies on specific primers to recognize target DNA sequences unique to the organism being detected, making it highly specific. Most molecular diagnostics uses the polymerase chain reaction (PCR), which involves repeated rounds of heating and cooling to make many copies of the target DNA, amplifying it so it can be detected. However the need for temperature cycling, and for sophisticated fluorescence-based systems to detect the amplified DNA, means that it is largely restricted to laboratory use. Moreover PCR is rather sensitive to inhibition by a number of common chemicals found in the environment and body fluids, requiring samples to be extensively purified before analysis.As a result the instruments required for PCR are vulnerable and relatively expensive to engineer. Molecular methods are therefore largely confined to large and expensive equipment within diagnostic laboratories requiring skilled operators. The limited equipment available for out-of-laboratory use is expensive, relatively large and unsuited to widespread application.The best solution to these problems is a recently developed approach based on amplification at a constant temperature, so-called isothermal amplification, coupled with a read-out of light as the specific target DNA sequence is amplified. This "bioluminescent assay in real-time" or BART results in a emission of a continuous light signal that reaches a peak at a time proportional to the amount of DNA present. BART was invented by the applicant and the CEO of the company started to commercialise such assays, Lumora Ltd. BART has been licensed by Lumora to the global partner 3M, who have commercialised assays for detecting food pathogens. This demonstrates the effectiveness and robustness of the approach.BART produces a light output that is simple and cheap to monitor using a camera chip or photodiodes in a solid-state device. This substantially reduces instrument costs and open up new applications for diagnostics and disease monitoring in resource-poor settings such as in the developing world, where there are extensive requirements for cheap molecular assays for disease diagnosis. A major challenge in molecular diagnostics remains the accurate measurement of the numbers of disease organisms (and hence target DNA molecules) in the sample. This can be done both by PCR and using BART, but the ability to accurately determine the number of molecules of the target becomes much more difficult below about 50 copies for both techniques. For certain diseases and situations, it is critical to be able to accurately measure the numbers of targets at this level. The proposed project is based on new methods discovered by the applicant and the company partner which are able to accurately determine the number of molecules down to a single molecule of target DNA. Moreover, remarkably they can distinguish between numbers of copies accurately in the range 1-10. The proposal is to test and develop these new approaches, and to use them with fluidic chips that would allow accurate measurement of numbers of molecules from 1 up to 100's of billions in a single set of assays carried out on a single plastic chip simultaneously- a so-called full dynamic range assay. Purpose designed algorithms will be developed and explored to analyse the data allowing the numbers to be quantified throughout the dynamic range using the most appropriate combination of methods for each part of the range.If successful, this would represent a breakthrough in molecular diagnostics with significant implications as a full dynamic range quantification method, which would see widespread application in research, medical diagnostics, disease monitoring, and environmental protection, with potential economic, health and societal benefits.

分子诊断是检测特定生物体、病原体或其他含有DNA的生物材料的最敏感技术，广泛用于世界各地的诊断实验室。它依赖于特异性引物来识别被检测生物体特有的靶DNA序列，使其具有高度特异性。大多数分子诊断使用聚合酶链反应（PCR），该反应涉及重复的加热和冷却循环，以产生靶DNA的许多拷贝，将其扩增，以便可以检测到。然而，需要温度循环和复杂的基于荧光的系统来检测扩增的DNA，这意味着它在很大程度上限于实验室使用。此外，PCR对环境和体液中的一些常见化学物质的抑制相当敏感，需要在分析前对样品进行大量纯化，因此PCR所需的仪器容易损坏，工程造价相对昂贵。因此，分子方法在很大程度上局限于需要熟练操作员的诊断实验室内的大型且昂贵的设备。这些问题的最佳解决方案是最近开发的一种基于恒温扩增的方法，即所谓的等温扩增，在扩增特定的靶DNA序列时结合光读出。这种“实时生物发光测定”或BART导致连续光信号的发射，该连续光信号在与存在的DNA的量成比例的时间达到峰值。BART是由申请人发明的，该公司的首席执行官Lumora Ltd.已将BART授权给全球合作伙伴3 M，后者已将检测食品病原体的检测方法商业化。这证明了该方法的有效性和鲁棒性。BART产生的光输出使用固态设备中的摄像头芯片或光电二极管进行监测是简单和便宜的。这大大降低了仪器成本，并为资源匮乏的环境（例如发展中国家）的诊断和疾病监测开辟了新的应用，这些国家对用于疾病诊断的廉价分子检测有广泛的需求。分子诊断中的主要挑战仍然是准确测量样品中疾病生物体（以及因此靶DNA分子）的数量。这可以通过PCR和使用BART来完成，但是对于这两种技术，在低于约50个拷贝时，准确确定靶分子数量的能力变得困难得多。对于某些疾病和情况，能够准确测量这一级别的目标数量至关重要。拟议的项目是基于申请人和公司合作伙伴发现的新方法，这些方法能够准确地确定目标DNA的单个分子的分子数量。此外，值得注意的是，它们可以在1-10的范围内准确地区分拷贝数。该提案是测试和开发这些新方法，并将它们与流体芯片一起使用，该流体芯片将允许在单个塑料芯片上同时进行的单组测定中精确测量从1到100亿的分子数量-所谓的全动态范围测定。将开发和探索专门设计的算法，以分析数据，从而使用最合适的方法组合在整个动态范围内对数字进行定量。如果成功，这将代表分子诊断学的突破，作为一种全动态范围定量方法，将在研究，医学诊断，疾病监测，和环境保护，具有潜在的经济、健康和社会效益。

项目成果

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

Convergent synthesis and optical properties of near-infrared emitting bioluminescent infra-luciferins.

• DOI: 10.1039/c6ra19541e
• 发表时间: 2017-01-16
• 期刊: RSC advances
• 影响因子: 3.9
• 作者: Anderson JC;Grounds H;Jathoul AP;Murray JAH;Pacman SJ;Tisi L
• 通讯作者: Tisi L

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

Bioluminescent detection of isothermal DNA amplification in microfluidic generated droplets and artificial cells.

• DOI: 10.1038/s41598-020-78996-7
• 发表时间: 2020-12-14
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Hardinge P;Baxani DK;McCloy T;Murray JAH;Castell OK
• 通讯作者: Castell OK

Additional file 1 of Lack of specificity associated with using molecular beacons in loop mediated amplification assays

附加文件 1 缺乏与在环介导扩增测定中使用分子信标相关的特异性

• DOI: 10.6084/m9.figshare.9210581
• 发表时间: 2019
• 作者: Hardinge P