Rapid point-of-care detection of Trichomonas Vaginalis

项目摘要 本项目的目标是演示一种用于检测患者的护理点(POC)的系统的可行性 整合了我们的专利病原体检测器的人阴道拭子样本中的阴道毛滴虫(TV) 基于物种特定的核酸(NA)序列的鉴定。探测器依赖于一种新的 实现低成本、无光学和无放大的机电信号转导机制 在超低浓度(~10-19M)下(例如，无PCR)检测DNA/RNA。快速发展的迫切需求依然存在 (分钟)，成本效益高，用于传染病诊断的POC NA检测设备，以便于及时， 知情的治疗和咨询，并避免潜在的有害行为，包括不适当的 抗生素处方。在早期的工作中，我们演示了用 灵敏度为98%，特异度为100%。滴虫病，最常见的可治愈的性传播疾病 在美国，这种疾病通常与新城疫和沙眼衣原体(CT)感染同时进行检测，而且 因此，这是我们要解决的一个合乎逻辑的目标。获得专利的探测器的一个关键特征是使用了肽核 酸(PNA)捕获探针，是不带电荷的聚酰胺类似物，具有相同的碱化学。 由于微珠-PNA偶联物被设计为电荷中性，它们在 直流电场的存在。然而，捕获目标时获得的大量负电荷 NA序列使杂化的共轭分子具有可移动性。微珠-PNA偶联物的电泳法 杂交靶NA到嘴部的直径较小的玻璃孔会导致孔洞的显著增加 电阻，从而导致测量的离子电流强劲、持续下降。非特定绑定的NA是 在孔口处的强电场中从珠子共轭中移除，导致没有持续的信号。 此外，相反的电渗流通过玻璃孔扫过PNA-珠联合物而不需要 杂交的目标远离毛孔口。通过这种方式，这种简单的电导测量设备提供了高度的 选择性(没有观察到的假阳性)，二元反应，发出目标NA存在或不存在的信号(和 相关病原体)。本课题主要围绕两个目标展开：1)电视残留物检测演示 来自加州大学洛杉矶分校临床微生物学实验室的阴道拭子样本具有竞争性高敏感性(≥95%) 和专一性(≥95%)和2)开发和可行性测试集成和健壮的 基于相对便宜的横向快速(&lt；5分钟)病原体检测的实验室原型装置 流动分析(LFA)格式。成功实现这些目标将产生一种设备概念，为 可制造的原型开发，能够以具有竞争力的准确性和可能性进行NG和TV测试 具有成本低、速度快、功耗大、健壮性强等显著优势。进一步的研究和开发工作是 设想将采样能力扩展到其他病原体，包括CT和其他复杂的样本介质 包括血和痰。

Project Summary The goal of this project is to demonstrate feasibility of a system for the point-of-care (POC) detection of Trichomonas vaginalis (TV) in human vaginal swab samples that integrates our 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) with sensitivity of ~98% and specificity of ~100%. Trichomoniasis, the most common curable, sexually transmitted disease in the US, is often tested for in parallel with NG and Chlamydia trachomatis (CT) infections and is therefore a 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 at 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 TV in remnant vaginal swab 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 laboratory prototype device for rapid (<5 mins) pathogen detection based on the relatively inexpensive lateral flow assay (LFA) format. Successful achievement of these Aims will result in a device concept ready for manufacturable prototype development that is capable of NG and TV 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, including CT, and to other complex sample media including blood and sputum.