Multiplexed pathogen identification via bead-based isothermal amplification in a low-cost microfluidic device

摘要： 呼吸道感染、腹泻疾病、败血症和尿路感染(UTIs)的治疗决定 与识别和区分许多可能的感染病原体有关(S)。核酸 (Nas)是病原体鉴定的有效生物标志物，但检测核酸序列通常需要 聚合酶链式反应的一些变种，需要复杂的仪器和训练有素的工作人员 只有在集中式实验室中才能找到。拟议项目的长期目标是发展一个点- 与CARE(POC)兼容的微流控设备用于9种不同尿路感染的DNA扩增和检测 病原体。拟议的项目将侧重于开发放大和检测组件，其中 今后的工作将与样品制备相结合。在这里提出的系统中，用户将添加 将提取的DNA放到一次性墨盒中，外部仪器将启动液体处理、热控制和 成像，结果将在1小时后公布。这种方法将使用等温核酸扩增， 它比PCR更适合PoC设置，因为它不需要热循环，从而减少了 价格昂贵、功能更强大的系统。然而，等温核酸扩增通常不适合于 更高阶复用(&gt；2或3个NA序列)。为了实现高阶复用，提出的方法将 结合了空间多路复用的优点，其中样本被分割并在不同的区域内放大 隔室，以及基于颜色的多路传输，其中使用独特的寡核苷酸检测探针的颜色来 识别一个扩增的序列。但是，我们将绕过这两种方法的限制，例如 由于样品稀释和用于区分激励的有限的滤波器空间而导致的空间多路复用中的灵敏度损失 以及彩色多路复用中的多个荧光团的发射。我们将使用克隆的等温核酸 在带有荧光编码微珠的油包水乳状液中进行放大，然后在 微通道。在目标1中，我们将建立所需的工艺来产生液滴-珠乳剂和 等温放大每个液滴中的Nas，导致扩增物结合到微珠上，然后是 打碎乳剂，并分离珠子进行成像。在目标2中，我们将设计和制造一个 适用于在护理地点使用以执行目标1中开发的过程的微流控设备。 3、我们将用从UTI病原体中提取的DNA来测试该设备，以验证该设备在识别方面的准确性 正确的病原体。在未来的工作中，我们将创造一个小型的仪器(&lt；1英尺3)，自动 以用户友好的集成格式启动流体处理、散热控制和成像组件 微流控装置就是在这里开发的。该装置还将与上游样品制备和 我们将用实际的临床尿路感染样本测试整个样本到答案的过程。

Summary: Treatment decisions for respiratory infections, diarrheal diseases, sepsis, and urinary tract infections (UTIs) are tied to the identification and differentiation of the many possible infection-causing pathogen(s). Nucleic acids (NAs) are effective biomarkers for pathogen identification, but detecting nucleic acid sequences typically requires some variation of the polymerase chain reaction (PCR), necessitating complex instrumentation and trained staff that are only found in centralized laboratories. The long-term goal of the proposed project is to develop a point- of-care (POC)-compatible microfluidic device for DNA amplification and detection of 9 different UTI-causing pathogens. The proposed project will focus on developing the amplification and detection components, which in future efforts will be integrated with sample preparation. In the herein proposed system, the user will add extracted DNA to a disposable cartridge, external instruments will actuate fluid handling, thermal control, and imaging, and the results will be available 1 hr later. This method will use isothermal nucleic acid amplification, which is more suitable for POC settings than PCR because it requires no thermocycling, resulting in less expensive and more robust systems. However, isothermal nucleic acid amplification is usually not suitable for higher order multiplexing (> 2 or 3 NA sequences). To achieve high-order multiplexing, the proposed method will combine the advantages of spatial multiplexing, where the sample is divided into and amplified within distinct compartments, and color-based multiplexing, where color of a unique oligonucleotide detection probe is used to identify an amplified sequence. However, we will circumvent the limitations of these two approaches, such as loss of sensitivity in spatial multiplexing due to sample dilution, and limited filter space to differentiate excitation and emission of multiple fluorophores in color multiplexing. We will use clonal isothermal nucleic acid amplification inside a water-in-oil emulsion with fluorescently encoded microbeads, followed by detection in a microchannel. In Aim 1, we will establish the required processes to generate the droplet-bead emulsions and isothermally amplify NAs within each droplet, resulting in amplicons bound to the microbeads, followed by breaking open the emulsion, and isolating the beads for imaging. In Aim 2, we will design and fabricate a microfluidic device appropriate for use at the point-of-care to execute the processes developed in Aim 1. In Aim 3, we will test the device with extracted DNA from UTI pathogens to validate the device's accuracy in identifying the correct pathogen. In future work, we will create a small compact instrument (< 1 ft3) that autonomously actuates the fluid handling, thermal control, and imaging components in an integrated user friendly format with the microfluidic device developed here. This device will also be coupled with upstream sample preparation and we will test the entire sample-to-answer process with actual clinical UTI samples.