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

项目摘要 该项目的目标是演示用于护理点的快速(≤5分钟)、灵敏系统的可行性 (POC)集成了我们的专利检测器的人阴道样本中人乳头瘤病毒(HPV)的检测 根据物种特定的核酸(NA)序列的鉴定，病原体的分类。该检测器依赖于 获得专利的机电信号传输机制，使低成本、免光学和 在超低浓度(~10-19M)下无扩增(例如，无聚合酶链式反应)检测DNA/RNA。迫切的需求 坚持使用快速(几分钟)、经济高效的POC NA检测设备进行传染病诊断，以便 促进及时、知情的治疗和咨询，并避免潜在的有害行动，包括 抗生素处方不当。在早期的工作中，我们演示了淋球菌的检测 (NG)的敏感性为98%，特异性为100%。我们目前正在收集类似的数据来检测 沙眼衣原体(CT)。由于HPV是最常见的性传播感染，也是宫颈发育缓慢的原因 对于癌症，这是我们为了促进挽救生命的癌前筛查而必须解决的一个合乎逻辑的目标。一项关键功能 我们的专利探测器之一是使用了肽核酸(PNA)捕获探针，这种探针是不带电荷的聚酰胺 类似于具有相同碱基化学的NAS。由于珠状-PNA偶联物被设计为带电 中性时，它们在直流电场存在的情况下不表现出电泳运动。然而， 在捕获目标NA序列时获得的大量负电荷使杂化的共轭 莫比尔县。微球-PNA结合物与杂交靶NA对较小直径口的电泳法 玻璃气孔导致气孔阻力显著增加，从而导致气孔阻力持续强劲下降。 测量到的离子电流。在强电中，非特定结合的NA从珠子共轭物中移除 毛孔口的磁场导致没有持续的信号。此外，相反的电渗流通过 玻璃孔将无杂化靶标的PNA-珠联结物从孔口扫走。以这样的方式， 这种简单的电导测量设备提供了高度选择性(没有观察到的假阳性)的二元响应 发出目标NA(和相关病原体)存在或不存在的信号。这个项目主要集中在三个方面 目的：1)检测≤10-19M(~60拷贝/毫升)培养上清液和人乳头瘤病毒感染者的高危型HPV mRNA 细胞数为~5-10个/毫升；2)基于横向流动分析(LFA)的集成原型的开发和测试 用于快速(≤5分钟)检测~5-10个/毫升的人乳头瘤病毒感染细胞的装置；以及3)演示 在残留的临床样本中检测到最高风险的HPV与批准的商业核糖核酸的符合率为95% 酸扩增试验(NAAT)。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 具有成本低、速度快、功耗低、坚固耐用等显著优势。

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.