Rapid point-of-care detection of Trichomonas Vaginalis

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， 人体阴道拭子样本中的阴道毛滴虫（TV），集成了我们获得专利的病原体检测仪 基于物种特异性核酸（NA）序列的鉴定。探测器依靠一本小说 这是一种机电信号转换机制，能够实现低成本、无光学和无放大 (e.g.,无PCR）检测超低浓度（~10-19 M）的DNA/RNA。迫切需要快速 （分钟），成本有效，用于传染病诊断的POC NA检测装置，以便于及时， 充分知情的治疗和咨询，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，我们证明了淋病奈瑟菌（NG）的检测与 敏感性约98%，特异性约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NG和沙眼衣原体（CT）感染并行检测， 因此是我们的目标专利检测器的一个关键特征是使用肽核酸 酸（PNA）捕获探针，其是共享相同碱基化学的NA的不带电荷的聚酰胺类似物。 由于珠粒-PNA缀合物被设计为电荷中性的，因此它们在电泳中不表现出电泳运动。 直流电场的存在。然而，在捕获目标时获得的大量负电荷 NA序列使杂交的缀合物具有移动的。珠粒-PNA缀合物的电泳 杂交靶NA到较小直径的玻璃孔的口导致孔的显著增加 电阻，从而导致测得的离子电流的强烈、持续下降。非特异性结合的NA是 在孔口处的强电场中从珠缀合物中除去，导致没有持续的信号。 此外，通过玻璃孔的相反电渗流扫过PNA-珠缀合物，而不产生任何干扰。 杂交目标远离毛孔。以这种方式，这种简单的电导测量装置给出了高度的 选择性（没有观察到假阳性），二元响应信号的存在或不存在的目标NA（和 相关病原体）。本项目主要有两个目的：1）演示残余物中TV的检测 来自UCLA临床微生物实验室的阴道拭子样本，具有竞争性高灵敏度（≥95%） 和特异性（≥95%）和2）开发和可行性测试的关键步骤，在一个综合的和强大的 用于快速（<5分钟）病原体检测的实验室原型设备， 流式分析（LFA）形式。这些目标的成功实现将导致一个设备的概念， 可制造的原型开发，能够进行NG和TV测试，具有竞争力的准确性， 具有成本、快速性、功率和鲁棒性的显著优点。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，以及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.