An Assay for Rapid and Sensitive Flu Virus Detection

描述(由申请人提供)：这项第一阶段SBIR提案的具体目标是确定一种新的检测技术的可行性，以快速和灵敏地检测流感病毒。具体地说，我们将确定手动化验是否快速(约15分钟)、灵敏(就TCID50单位/毫升而言，至少是FDA批准的抗原检测的100倍或更好)、重复性(CV&lt；15%)和特异性(浓度高达106CFU/毫升的潜在干扰细菌物种没有假阳性结果)。建议的研究有五个具体目标，包括： 1.荧光素-4，7-二甲基-O-神经氨酸底物的合成、纯化及鉴定我们的目标是获得至少1克的高纯度底物(通过高效液相色谱检测纯度为98%)。 2.检测混合物的配方--第D2节。我们将准备一种检测混合物，其中包含检测流感病毒神经氨酸酶所需的所有试剂。检测混合物将在一次性使用的检测管中冷冻干燥。 3.测试优化--第D3节 4.分析业绩的评价--第D4节。将对该分析的分析性能进行评估。我们将确定10多种人类甲型和乙型流感病毒株的分析敏感性。 5.使用人体样本进行比较研究和初步评估--第D5节。我们将使用FDA批准的基于抗原的检测和RT-PCR检测进行比较研究。此外，我们还将对从受试者身上采集的一些样本进行检测。 拟议的第一期SBIR项目将评估一种新的检测技术的可行性，以快速和灵敏地检测流感病毒。该技术通过使用母液简化了检测过程，该母液实现了一步检测，即将样品添加到母液中，然后进行光信号测量，所有这些都在相同的检测管中进行。整个过程需要10-15分钟。拟议的流感病毒测试的成功开发将为流感快速诊断提供更好的替代方案，从而显著提高我们应对大流行间流感和潜在大流行流感的医疗能力。

DESCRIPTION (provided by applicant): The specific aims of this Phase I SBIR proposal are to determine the feasibility of a novel detection technology for rapid and sensitive detection of influenza virus. Specifically, we will determine if the manual assay is rapid (about 15 minutes), sensitive (as least 100 times as good as, or better than, an FDA approved antigen test in terms of TCID50 units/mL), reproducible (with CV<15%) and specific (without false positive results from potentially interfering bacterial species at concentrations of upto106 cfu/mL). There are five specific aims in the proposed studies, including: 1. Synthesis, purification and qualification of luciferin - 4,7-di-methyl-O-neuraminic acid substrate - Section D1. Our goal is to obtain at least 1 gram of highly purified substrate (>98% purity by HPLC). 2. Formulation of the detection mix - Section D2. We will prepare a detection mix that contains all necessary reagents for detection of flu viral neuraminidase. The detection mix will be lyophilized in a single-use detection tube. 3. Assay optimization - Section D3 4. Evaluation of analytical performance - Section D4. Analytical performance of the assay will be evaluated. We will determine the analytical sensitivity for more than 10 strains of human influenza A and human influenza B. 5. Comparison studies and preliminary evaluation using human samples - Section D5. We will perform comparison studies using FDA-approved antigen-based test and RT-PCR assay. In addition, we will test some samples collected from human subjects. PUBLIC HEALTH RELVANCE The proposed Phase I SBIR project will evaluate the feasibility of a novel detection technology for rapid and sensitive detection of influenza virus. The technology simplifies the detection process by using a master mix which enables one-step detection, i.e., addition of a sample to the master mix followed by light signal measurement, all performed in the same detection tube. The entire process takes 10-15 minutes. Successful development of the proposed influenza virus test will provide a better alternative for rapid flu diagnosis, thereby significantly improving our medical capability for interpandemic influenza and potential pandemic influenza.