Rapid point-of-care detection of Chlamydia trachomatis in urine

项目摘要 该项目的目标是证明系统的床旁（POC）检测的可行性， 人体尿液样本中的沙眼衣原体（CT），集成了一种专利的病原体检测器， 物种特异性核酸（NA）序列的鉴定。探测器依靠一种新型的机电 信号转导机制，其能够实现低成本、无光学和无扩增（例如，无PCR） 检测超低浓度（~10 - 19 M）的DNA/RNA。迫切需要快速（分钟）、低成本 用于传染病诊断的有效的POC NA检测设备，以便于及时、充分了解 治疗和咨询，并避免潜在的有害行为，包括不适当的处方， 抗生素在早期的工作中，我们证明了淋病奈瑟菌（NG）的检测，这是CDC的前三名 公共卫生威胁，灵敏度约98%，特异性约100%。衣原体是最常见的 在美国，这种疾病最常与NG同时检测，因此是我们下一个最合理的目标 来解决。专利检测器的一个关键特征是使用肽核酸（PNA）捕获探针， 是共享相同基础化学的NA的不带电聚酰胺类似物。由于珠粒-PNA缀合物是 设计为电荷中性，它们在直流电存在下不表现出电泳运动。 领域然而，在捕获靶NA序列时获得的大量负电荷使得靶NA序列的电荷减少。 杂化共轭物是移动的。具有杂交的靶NA的珠粒-PNA缀合物电泳至口腔 较小直径的玻璃孔导致孔阻力的显著增加，从而导致强的， 测量的离子电流持续下降。非特异性结合的NA从微珠缀合物中除去。 孔口中的强电场导致没有持续的信号。此外，相反的电渗流 通过玻璃孔将没有杂交靶的PNA-珠缀合物扫离孔口。在 通过这种方式，这种简单的电导装置给出了高度选择性（没有观察到假阳性）、二进制 反应信号的存在或不存在的目标NA（和相关的病原体）。该项目重点 两个目的：1）证明来自UCLA临床试验室的残余尿液样本中的CT检测 具有竞争力的高灵敏度（≥ 95%）和特异性（≥ 95%）的微生物实验室和2）开发 和可行性测试的关键步骤，在一个集成的和强大的原型装置，快速（<5分钟）细菌 基于使用磁珠的检测，最大限度地减少了对多个腔室、泵和阀的需求。 这些目标的成功实现将导致一个设备的概念准备原型开发， 能够以具有竞争力的准确度进行NG和CT测试，并且可能具有成本，快速性， 功率和鲁棒性。预计将开展进一步的研究和开发工作，以扩大取样能力 其它病原体，包括微生物和病毒，以及其它复杂的样品介质，包括血液和痰。

Project Summary The goal of this project is to demonstrate feasibility of a system for the point-of-care (POC) detection of Chlamydia trachomatis (CT) in human urine samples that integrates a patented detector of pathogens based on identification of species-specific, nucleic acid (NA) sequences. The detector relies on a novel electromechanical signal transduction mechanism that enables the low-cost, optics-free and amplification-free (e.g., no PCR) detection of DNA/RNA at ultralow concentration (~10-19 M). A compelling need persists for rapid (minutes), cost effective, POC NA detection devices for infectious disease diagnostics so as to facilitate prompt, well-informed therapies and counseling and to avoid potentially harmful actions including the inappropriate prescription of antibiotics. In earlier work, we demonstrated the detection of Neisseria gonorrhoeae (NG), a CDC top-three public health threat, with sensitivity of ~98% and specificity of ~100%. Chlamydia, the most common notifiable disease in the US, is most often tested for in parallel with NG and is therefore the next most logical target for us to address. A key feature of the patented detector is the use of peptide nucleic acid (PNA) capture probes, which are uncharged polyamide analogs to NAs that share the same base chemistry. Since bead-PNA conjugates are designed to be charge neutral, they do not exhibit electrophoretic movement in the presence of a DC electric field. However, the substantial negative charge acquired upon capture of a target NA sequence makes the hybridized conjugate mobile. Electrophoresis of the bead-PNA conjugate with hybridized target NA to the mouth of a smaller diameter glass pore causes a significant increase in pore resistance, thereby resulting in a strong, sustained drop in measured ionic current. Nonspecifically bound NA is removed from the bead conjugate in the strong electric field in the pore mouth resulting in no sustained signal. Further, the opposing electroosmotic flow through the glass pore sweeps PNA-bead conjugates without hybridized target away from the pore mouth. In such a way, this simple conductometric device gives a highly selective (no observed false positives), binary response signaling the presence or absence of the target NA (and associated pathogen). This project focuses on two Aims: 1) Demonstration of the detection of CT in remnant urine samples from the UCLA Clinical Microbiology Laboratory with competitively high sensitivity (≥95%) and specificity (≥95%) and 2) Development and feasibility testing of key steps in an integrated and robust prototype device for rapid (<5 mins) bacterial detection based on the use of magnetic beads that minimizes the need for multiple chambers, pumps and valves. Successful achievement of these Aims will result in a device concept ready for prototype development that is capable of NG and CT testing with competitive accuracy and likely with significant advantages of cost, rapidity, power and robustness. Further research and development work is envisioned to expand sampling capabilities to other pathogens, both microbial and viral, and to other complex sample media including blood and sputum.