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

分子诊断是检测含有DNA的特定生物体、病原体或其他生物材料的最灵敏的技术，在全世界的诊断实验室中得到广泛应用。它依靠特定的引物来识别被检测生物体特有的目标DNA序列，使其具有高度特异性。大多数分子诊断都使用聚合酶链反应（PCR），这种方法包括反复加热和冷却，以复制目标DNA的许多副本，将其放大，以便检测到。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仅限于实验室使用。此外，PCR对环境和体液中发现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广泛纯化。因此，聚合酶链反应所需的仪器很脆弱，而且设计起来相对昂贵。因此，分子方法主要局限于诊断实验室中需要熟练操作人员的大型昂贵设备。可用于实验室外使用的有限设备昂贵，相对较大，不适合广泛应用。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，在扩增特定目标DNA序列时加上光读出。这种“实时生物发光测定”或BART的结果是连续光信号的发射，该信号在与存在的DNA数量成正比的时间达到峰值。BART是由申请人发明的，该公司的首席执行官Lumora Ltd.开始将这种检测方法商业化。Lumora已将BART授权给全球合作伙伴3M，后者已将用于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产生的光输出既简单又便宜，可以使用固态设备中的摄像芯片或光电二极管进行监控。这大大降低了仪器成本，并在资源贫乏的环境中开辟了诊断和疾病监测的新应用，例如在发展中国家，那里对用于疾病诊断的廉价分子分析有广泛的需求。分子诊断的一个主要挑战仍然是准确测量样本中疾病生物体（以及目标DNA分子）的数量。这可以通过PCR和BART来实现，但是对于这两种技术来说，精确确定目标分子数量的能力在大约50个拷贝以下变得更加困难。对于某些疾病和情况，能够准确衡量这一水平上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的分子数量，直至单个分子。此外，值得注意的是，它们可以准确地区分1-10个范围内的拷贝数。他们的建议是测试和开发这些新方法，并将它们与流体芯片一起使用，这样就可以在一个塑料芯片上同时进行一组分析，精确测量从1到1000亿的分子数量，即所谓的全动态范围分析。将开发和探索目的设计的算法来分析数据，允许在整个动态范围内使用最合适的方法组合对范围的每个部分进行量化。如果成功，这将是分子诊断学的一个突破，作为一种全动态范围定量方法具有重要意义，将广泛应用于研究、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。

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.