Biosensor Assays for Diagnosis of Chronic HCV Infection

描述(申请人提供)：此第一阶段SBIR申请是响应卫生部的号召，要求进行新的丙型肝炎病毒RNA定性检测，以诊断患者的慢性丙型肝炎病毒感染。现在需要的是与目前的分子检测方法一样可靠但成本更低、资源要求更高的丙型肝炎病毒RNA检测方法。拟议用于研发的新测试包括快速样品分离、创新的信号放大和使用新的专有Alderon生物传感器进行检测。本研究的创新之处在于通过开发集成的RNA分离和先进的电化学(ESystem)检测技术，建立了一种新的、廉价、低成本的丙型肝炎病毒RNA检测方法，其可行性和性能特征将在三个特定目标的研究中进行研究。在目标1中，我们将尝试演示一种简单的模式，使生物体液样本中的病毒RNA可用于我们下游的eSystem分析。在目标2中，我们将使用一种获得专利的信号放大方法(PESA)和一种新的毛细管流动设计来研究创新的丙型肝炎病毒RNA检测，该设计用于先进、低成本的生物传感器，这些传感器产生与目标相关的电流，通过我们开发的eSystem读取器/监视器进行测量。这些检测将采用直接检测目标特定的酶标记以获得高灵敏度，而不需要酶增强检测通常所需的洗涤步骤。我们的低成本读取器将提供受控的电脉冲，并监测目标特定酶标签产生的电流。AIM 2的里程碑将是确定这种新的丙型肝炎病毒RNA检测模式是否具有灵敏的电响应，准确检测到500IU/ml。我们的目标是一种生物传感器系统，其性能与现有FDA批准的分支探针(BProbe)检测方法相同，但适用于在护理点广泛使用。在这些Aim 2研究中，我们将使用装甲丙型肝炎病毒RNA标准和模拟临床标本，其中包括添加到血清和血浆样本中的丙型肝炎病毒。在目标3中，我们将用临床标本验证Alderon丙型肝炎病毒RNA定性eSystem分析。实现这些目标将表明开发新的eSystem定性丙型肝炎病毒RNA测试的可行性，这种测试在临床上与商业上可用的FDA批准的慢性丙型肝炎病毒感染确证测试一样有用和可靠，但成本低得多，适合在资源有限的社区使用。我们预计，这种定性的丙型肝炎病毒RNA检测的新方法将被用作临床诊断和作为研究丙型肝炎病毒慢性感染的有价值的研究工具。在该项目中开发的拟议的eSystem技术将产生很大的影响，因为它将为更多的感染者带来更低成本的丙型肝炎病毒检测。项目成功的其他影响将来自使用基本技术来开发其他RNA病毒的新测试，例如艾滋病毒。

DESCRIPTION (provided by applicant): This Phase I SBIR application is submitted in response to the call by DHHS for new tests for qualitative measurement of HCV RNA for diagnosis of chronic HCV infection in patients. The need is for HCV RNA assays that are as reliable but less expensive and resource-demanding than current molecular assays. The new test proposed for research and development consists of rapid sample isolation, innovative signal amplification, and detection with a new proprietary Alderon biosensor. The innovation in this research lies in the creation of a new, inexpensive, low-cost, assay for HCV RNA by development of integrated RNA isolation and advanced electrochemical (eSystem) detection, whose feasibility and performance characteristics will be investigated in research with three specific aims. In Aim 1, we will seek to demonstrate a simple mode of making the viral RNA in biofluid samples available for our downstream eSystem assays. In Aim 2, we will investigate innovative HCV RNA detection using a patented signal amplification method (PESA) and a new capillary flow design for advanced, low-cost biosensors that generate target-dependent electrical currents that are measured with an eSystem reader/monitor developed by us. The assays will employ direct detection of target-specific enzyme labels for high sensitivity, without the wash steps usually required for enzyme-enhanced assays. Our low-cost readers will deliver controlled electrical pulses and monitor currents generated by the target-specific enzyme labels. The milestone for Aim 2 will be to determine whether this new mode of HCV RNA detection has a sensitive electrical response with accurate detection down to 500 IU/mL. The objective is a biosensor system with performance equal to that of existing FDA-approved, branched-probe (bProbe) assays, yet practical for wide spread use at point of care. In these Aim 2 studies, we will use Armored HCV RNA standards and mock clinical specimens consisting of the hepatitis C virus spiked into serum and plasma samples. In Aim 3, we will validate the Alderon HCV RNA Qualitative eSystem Assay with clinical specimens. Accomplishing these aims will show the feasibility of developing new eSystem qualitative HCV RNA tests that are as clinically useful and reliable as commercially available, FDA-approved tests for confirmatory testing of chronic HCV infections but at much lower cost and suitable for use in resource-limited communities. We anticipate that this new approach to qualitative HCV RNA testing will find use both as a clinical diagnostic and as a valuable research tool for the study of HCV chronic infections. The proposed eSystem technology to be developed in this project will have a high impact, as it brings lower-cost HCV testing to many more infected individuals. Additional impacts of project success will result from use of the basic technology to develop new tests for other RNA viruses, such as HIV.