SBIR PA22-176 - Rapid Point-of-Care Detection of Human Papillomavirus

SBIR PA22-176 - 人乳头瘤病毒的快速护理点检测

• 批准号: 10761537
• 负责人: Zhenrong Zheng
• 金额: $ 27.58万
• 依托单位: ELECTRONUCLEICS, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2024-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10761537
• 关键词: SBIR; PA22; 176; Rapid; Point

Project Summary The goal of this project is to demonstrate feasibility of a rapid (≤5 mins), sensitive system for the point-of-care (POC) detection of human papillomavirus (HPV) in human vaginal samples that integrates our patented detector of pathogens based on identification of species-specific, nucleic acid (NA) sequences. The detector relies on a patented 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%. We currently are gathering similar data for detection of Chlamydia trachomatis (CT). Since HPV is the most common STI and the cause of slowly developing cervical cancers, it is a logical target for us to address in order to facilitate life-saving precancer screening. A key feature of our 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 three Aims: 1) Detection of highest risk HPV mRNA in media at ≤10-19 M (~60 copies/mL) and of HPV-infected human cells at ~5-10 cell/mL; 2) Development and testing of an integrated, lateral flow assay (LFA)-based prototype device for rapid (≤5 mins) detection of HPV-infected human cells at ~5-10 cells/mL; and 3) Demonstration of highest risk HPV detection in remnant clinical samples in 95% concordance with an approved commercial nucleic acid amplification test (NAAT). Successful achievement of these Aims will result in a device concept ready for manufacturable prototype development that is capable of HPV testing with competitive accuracy and with significant advantages of cost, rapidity, low power and robustness.

项目摘要 本项目的目标是证明快速（≤5分钟）、灵敏的床旁系统的可行性 (POC)检测人类阴道样本中的人乳头瘤病毒（HPV），集成了我们的专利检测器 基于物种特异性核酸（NA）序列的鉴定的病原体。探测器依靠一个 专利的机电信号转换机制，使低成本，无光学， 无扩增（例如，无PCR）检测超低浓度（~10-19 M）的DNA/RNA。亟需 持续用于传染病诊断的快速（分钟）、成本有效的POC NA检测设备，以便 促进及时、知情的治疗和咨询，避免潜在的有害行为，包括 抗生素处方不当。在早期的工作中，我们证明了淋病奈瑟菌的检测 (NG)敏感性约98%，特异性约100%。我们目前正在收集类似的数据， 沙眼衣原体（CT）。由于HPV是最常见的性传播感染， 因此，它是我们解决的一个合乎逻辑的目标，以促进挽救生命的癌前筛查。一个关键特征 我们的专利检测器的一个优点是使用肽核酸（PNA）捕获探针， 具有相同碱基化学的NA类似物。由于珠粒-PNA缀合物被设计成是电荷的， 中性，它们在直流电场存在下不表现出电泳运动。但 在捕获靶NA序列时获得的大量负电荷使杂交的缀合物 移动的。将具有杂交的靶NA的珠粒-PNA缀合物电泳至较小直径的口 玻璃孔引起孔阻力的显著增加，从而导致玻璃孔的强度的持续下降。 测量离子电流。非特异性结合的NA在强电场中从珠缀合物中除去。 毛孔口处的场导致没有持续的信号。此外，通过微电极的相反电渗流可以被抑制。 玻璃孔将没有杂交靶的PNA-珠缀合物从孔口中扫走。通过这种方式， 这种简单的电导装置给出了高度选择性（没有观察到假阳性）的二元响应 发出存在或不存在靶NA（和相关病原体）的信号。本项目重点关注三个 目的：1）检测培养基中≤10-19 M（~60拷贝/mL）的高危HPV mRNA和HPV感染的人 约5-10个细胞/mL的细胞; 2）开发和测试基于侧向流测定（LFA）的集成原型 用于以约5-10个细胞/mL快速（≤5分钟）检测HPV感染的人细胞的装置;以及3） 剩余临床样本中最高风险HPV检测结果与获批的商业核酸检测结果一致率为95% 酸性扩增试验（NAAT）。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，能够以具有竞争力的准确性进行HPV检测， 成本、快速性、低功耗和鲁棒性的显著优点。