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

快速即时检测尿液中的沙眼衣原体

基本信息

批准号：
10260070
负责人：
Sam Bae
金额：
$ 14.99万
依托单位：
ELECTRONUCLEICS, INC.
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-22 至 2022-10-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10260070
关键词：
Achievement Acute Address Adsorption Antibiotics Base Sequence Beds Binding Biological Assay Blood Caliber Cells Centers for Disease Control and Prevention (U.S.)Charge Chemicals Chemistry Chlamydia Chlamydia trachomatis Clinical Clinical Microbiology Collaborations Communicable Diseases Complex Counseling Cytolysis DNA Detection Development Devices Diagnostic Disease Drops Electrophoresis Ensure Event Filtration Glass Goals Gold Gonorrhea Human Hybrids Laboratories Legal patent Magnetism Measures Microfluidics Movement Neisseria gonorrhoeae Nucleic Acid Amplification Tests Nucleic Acid Hybridization Nucleic Acids Nucleotides Nylons Optics Oral cavity Particulate Matter Peptide Nucleic Acids Performance Phase Point-of-Care Systems Polymerase Polystyrenes Process Protocols documentation Public Health Pump RNA Reaction Reagent Resistance Ribosomal RNA Sampling Scheme Sensitivity and Specificity Signal Transduction Specificity Sputum System Technology Testing Time Urine Viral Work analog base comparative cost cost effective data acquisition design detection platform detector electric field feasibility testing inhibitor/antagonist magnetic beads magnetic field microbial novel nucleic acid detection nucleic acid purification pathogen point of care point-of-care detection point-of-care diagnostics polymerization prototype research and development response sensor sensor technology success

项目摘要

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.

项目概要该项目的目标是证明用于即时护理 (POC) 检测系统的可行性人体尿液样本中的沙眼衣原体 (CT)，集成了基于专利的病原体检测器该检测器依赖于一种新型机电技术来识别物种特异性核酸 (NA) 序列。信号转导机制可实现低成本、无光学和无扩增（例如无需 PCR）超低浓度（~10-19 M）DNA/RNA 的检测仍然存在对快速（分钟）、成本的迫切需求。用于传染病诊断的有效的 POC NA 检测设备，以便于及时、信息灵通治疗和咨询，并避免潜在的有害行为，包括不适当的处方在早期的工作中，我们展示了 CDC 排名前三的淋病奈瑟菌 (NG) 的检测。公共卫生威胁，敏感性约为 98%，特异性约为 100%，是最常见的需通报的衣原体。在美国，这种疾病最常与 NG 并行检测，因此是我们下一个最合理的目标该专利检测器的一个关键特征是使用肽核酸 (PNA) 捕获探针。是不带电荷的 NA 类似物，具有相同的基础化学性质，因为珠子-PNA 缀合物是设计为电荷中性，它们在存在直流电的情况下不会表现出电泳运动然而，捕获目标 NA 序列后获得的大量负电荷使得将带有杂交目标 NA 的珠子-PNA 缀合物电泳至口腔。较小直径的玻璃孔会导致孔阻力显着增加，从而产生强大的、测量的离子电流持续下降，非特异性结合的 NA 从珠缀合物中去除。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目标的 PNA-珠缀合物从孔口中扫走。通过这种方式，这种简单的电导测量装置提供了高度选择性（没有观察到误报）、二元响应信号表明目标 NA（和相关病原体）是否存在。两个目标：1) 演示在加州大学洛杉矶分校临床实验室的残余尿样中进行 CT 检测具有竞争性高灵敏度（≥95%）和特异性（≥95%）的微生物学实验室和2）开发以及对快速（<5 分钟）细菌的集成且稳健的原型设备中的关键步骤进行可行性测试基于使用磁珠的检测，最大限度地减少对多个腔室、泵和阀门的需求。成功实现这些目标将导致为原型开发做好准备的设备概念：能够以具有竞争力的精度进行 NG 和 CT 测试，并可能具有成本、快速、预计将进行进一步的研究和开发工作以扩大采样能力。其他病原体（微生物和病毒）以及其他复杂的样本介质（包括血液和痰）。