Next Generation of Ultra-Sensitive DNA Diagnostics for Gastric Infections

多年来，我们一直致力于检测与传染病有关的DNA序列的方法，重点是使用功能化纳米颗粒和表面增强拉曼散射。这项工作已经产生了许多不同的检测方法，目前正在通过雷尼绍诊断有限公司进行开发。现在这些检测方法正在进入最终用户手中，市场反馈建议可以对检测方法进行改进，使其在行业最新发展中更具竞争力，并提高非专业人员的易用性。这个博士学位的目的是研究以一种多路和简单的方式检测指示胃部感染的特定DNA序列，而不需要像大多数商业检测方法中所使用的那样对目标进行PCR扩增。转向基于无聚合酶链式反应的分析的挑战主要是在灵敏度要求方面，其中聚合酶链式反应极大地增加了检测的机会，而且在通过使用聚合酶链式反应控制DNA长度而将目标与探针杂交的简单性方面，这项建议的目的是开发一种新的分析方法，在不需要任何酶步骤的情况下检测胃感染的临床相关范围(少于107拷贝)的DNA序列。其目的是：1.研究使用固定相如硝化棉固定DNA的探针序列，以捕获目标DNA并洗去多余的DNA。这还包括添加DNA功能化纳米颗粒，我们将使用它们与表面捕获的靶序列杂交。我们将通过使用纳米颗粒组装方法在灵敏度上进一步飞跃，我们已经通过DNA杂交证明了这种方法是有效的。这将只发生在附着在表面DNA序列上的纳米颗粒上。在测试了上述系统后，我们将转向临床样本，并使用包含模型测试系统中未找到的多种成分的临床样本来评估该分析格式的性能。创建一种用于验证和确认这种方法的工作装置，用于超灵敏的DNA检测，这应该可以在样品采集后不到1小时内完成。

We have been working on methodologies to detect DNA sequences relating to infectious disease for a number of years with a focus on using functionalised nanoparticles and surface enhanced Raman scattering. This work has produced a number of different assay approaches which are now under development through Renishaw Diagnostics Ltd. Now that these assays are moving into the hands of the end user, the market feedback suggests that improvements can be made to the assays to make them more competitive with the latest developments in the sector and also to improve the ease of use for non-specialists. This PhD studentship is designed to investigate the detection of specific DNA sequences indicative of gastric infections in a multiplexed and simple manner without the need for PCR amplification of the targets as is used in a majority of commercially available assays. The challenge of moving to a PCR free based assay is mainly in terms of the sensitivity requirements where PCR greatly increases the chance of detection but also in terms of the simplicity of hybridisation of the target with the probes through the control of the length of the DNA using PCR The aim of this proposal is to develop a new assay to detect DNA sequences in a clinically relevant range (less than 107 copies) for gastric infections without any enzyme steps. The objectives are:1. To investigate the use of a stationary phase such as nitrocellulose for the immobilisation of a probe sequence of DNA to capture the target DNA and wash away excess. This will also involve the addition of DNA functionalised nanoparticles which we will use to hybridise to the captured target sequence on the surface. We will take a further leap in sensitivity by using a nanoparticle assembly approach which we have already shown to work through DNA hybridisation. This will take place only on the nanoparticles attached to the DNA sequence on the surface.2. Having tested the above system we will move towards clinical samples and evaluate the performance of this assay format with clinical samples which contain a multitude of components not found in the model test systems.3. To create a working device for verification and validation of this approach for use in ultrasensitive DNA detection which should be possible in less than 1 hour from sample collection.