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

总结： 呼吸道感染、呼吸道疾病、败血症和尿路感染（UTI）的治疗决策 与许多可能引起感染的病原体的鉴定和区分有关。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 聚合酶链反应（PCR）的一些变化，需要复杂的仪器和训练有素的工作人员 只有在中心实验室才能找到。该项目的长期目标是开发一个点- 用于DNA扩增和检测9种不同UTI引起的 病原体拟议项目将侧重于开发扩增和检测组件， 今后的工作将与样品制备相结合。在本文提出的系统中，用户将添加 提取的DNA到一次性盒中，外部仪器将启动流体处理，热控制， 成像，结果将在1小时后提供。该方法将使用等温核酸扩增， 这比PCR更适合于POC设置，因为它不需要热循环， 更昂贵和更强大的系统。然而，等温核酸扩增通常不适用于扩增。 高阶复用（> 2或3个NA序列）。为了实现高阶复用，所提出的方法将 联合收割机结合了空间多路复用的优点，在空间多路复用中，样品被分成不同的部分并在不同的部分内被放大。 隔室和基于颜色的多路复用，其中独特的寡核苷酸检测探针的颜色用于 识别扩增序列。但是，我们将绕过这两种方法的限制，例如 由于样品稀释，空间多路复用中的灵敏度损失，以及用于区分激发的有限滤波器空间 以及在颜色多路复用中发射多个荧光团。我们将使用克隆等温核酸 在具有荧光编码的微珠的油包水乳液内进行扩增，然后在 微通道在目标1中，我们将建立产生液滴-珠粒乳液所需的工艺， 等温扩增每个液滴内的NA，产生与微珠结合的扩增子，然后 将乳液破碎，并分离珠粒用于成像。在目标2中，我们将设计和制造一个 微流体装置适合在护理点使用，以执行目标1中开发的过程。在Aim中 3，我们将用从UTI病原体中提取的DNA测试该设备，以验证该设备在识别UTI病原体方面的准确性。 正确的病原体在未来的工作中，我们将创建一个小型紧凑的仪器（< 1立方英尺）， 以集成的用户友好格式驱动流体处理、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UTI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.