Integrated molecular diagnostic system for the point-of-care

描述(由申请人提供)：这个STTR-AT-NIAID旨在开发一个基于凯瑟琳·克拉珀里奇在波士顿大学的实验室所做的研究的综合核酸系统。BU Lab-on-a-Check包括微型固相萃取(&lt；SPE)柱、用于通过微通道控制流体运动的翻板阀和疏水通风口，以及用于实验复制和控制反应的多个反应室。克拉珀里奇博士的实验室已经成功地使用BioHelix的等温放大过程在芯片中进行了核酸放大。Biohelix的专利技术被称为解旋酶依赖扩增(HDA)。它使用DNA解旋酶在65℃恒定温度下的指数扩增过程中分离DNA链，像聚合酶链式反应(PCR)一样，HDA分析使用竞争性内部对照(即，将已知浓度的模板DNA添加到原始样本中)，该内部对照可以通过与分析物相同的引物进行扩增，但可以单独检测；因此，我们可以在直接的临床样本中检测放大抑制剂。第一阶段的目标是：1)修改BU微流控设备的设计，以纳入横向流动条，作为使用肉眼检测扩增产物的手段；以及2)证明使用Aim 1的设备进行综合测试的可行性，使用CT和NG在每毫升尿液中添加104%的CT和NG，以建立在包括横向流动条的改良BU芯片中进行综合分析的概念证据。我们第二阶段的具体目标将是开发横向流动读取器子系统，将其与BU仪器集成，以构建预商用外观/工程样样机，简化芯片设计和制造，以便进行中试规模制造，并在BioHelix/Quidel实施一次性设备的中试规模制造。在这个阶段，启根是最有可能的阅读器制造商。在第二阶段结束时，我们应该为新检测系统的临床验证做好准备。 公共卫生相关性：这一第一阶段STTR-AT-NIAID项目寻求概念验证项目将侧重于最丰富的性传播疾病(STD)病原体：沙眼衣原体(CT)和淋球菌(NG)。科学文献清楚地表明，分子检测是检测CT和NG最敏感的手段，分子CT/NG高通量筛查市场目前的价值超过3亿美元/年。此外，CDC敦促性病诊所对进行POC检测的患者进行检测，如果医护人员怀疑这些患者不太可能回到STD诊所了解检测结果。不幸的是，没有护理点(POC)CT NG分子检测，而且现有的POC分子检测系统(如GeneXpert)太昂贵，无法在性病诊所使用。这项拟议的项目将寻求弥补这一缺陷。我们建议使用凯瑟琳·克拉珀里奇博士在波士顿大学(BU)的实验室开发的设计来开发一个低成本的POC分子诊断系统。尽管目前的BU一次性可以执行我们专有的等温放大反应，但它不允许低成本、无需仪器地检测扩增产物；即，可以使用荧光显微镜检测产物的形成，但这不是一个商业上可行的选择。我们设想的第一阶段的设备将包括一个横向流动条，作为一种通过简单的肉眼检查来检测核酸扩增产物的存在或不存在的手段。Biohelix在使用基于横向流动的检测开发分子测试方面拥有经验。

DESCRIPTION (provided by applicant): This STTR-AT-NIAID seeks to develop an integrated nucleic acid system based on research done by Catherine Klapperich's laboratory at Boston University. The BU lab-on-a-chip includes a micro solid phase extraction (<SPE) column, flap valves and hydrophobic vents to gate fluid movements through micro channels, and multiple reaction chambers for experimental replicates and control reactions. Dr. Klapperich's laboratory has successfully performed nucleic acid amplifications in the chip using BioHelix's isothermal amplification process. Biohelix's proprietary technology is called helicase-dependent amplification (HDA). It uses DNA helicases to separate DNA strands during exponential amplification at a constant temperature of 65:C. Like the polymerase chain reaction (PCR), HDA assays use a competitive internal control (i.e., a template DNA of known concentration spiked into the raw sample) that can be amplified by the same primers as the analyte, but detected separately; hence allowing us to detect amplification inhibitors in direct clinical samples. The objectives of Phase I are to: 1) modify the BU microfluidic device design to incorporate a lateral flow strip as a means of detecting amplification products using the naked eye; and 2) demonstrate the feasibility of performing integrated tests using the device from aim 1 using CT and NG spiked at 104 in 1 mL urine to establish proof of concept for integrated assays performed in a modified BU chip that includes a lateral flow strip. Our specific aims for Phase II will be to develop a lateral flow reader sub-system, integrate it with the BU instrument to build a pre-commercial looks-like / works-like prototypes, simplify chip design and fabrication to allow for pilot scale manufacturing, and implement a pilot scale manufacturing for the disposable at BioHelix / Quidel. At this stage Qiagen is the most likely manufacturer for the reader. At the conclusion of Phase II, we should be ready for clinical validation of the new assay system. PUBLIC HEALTH RELEVANCE: This Phase I STTR-AT-NIAID project seeks proof-of-concept project will focus on the most abundant sexually transmitted disease (STD) pathogens: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). The scientific literature clearly shows that molecular testing is the most sensitive means of detecting CT and NG and the molecular CT/NG high throughput screening market is currently valued at over $300M/year. Moreover, CDC urges STD clinics to test patients with POC tests if health care workers suspect these patients are unlikely to return to the STD clinic to learn the results of the test. Unfortunately, there are no point-of-care (POC) CT NG molecular tests, and existing POC molecular testing systems like the GeneXpert are too costly for use in STD clinics. This proposed project would seek to remedy to this short coming. We propose to develop a low-cost POC molecular diagnostic system using a design developed by Dr. Catherine Klapperich's laboratory at Boston University (BU). Although the current BU disposable can perform our proprietary isothermal amplification reactions, it does not allow for low cost, instrument-free detection of amplification products; i.e., a fluorescence microscope can be used to detect product formation but this is not a commercially viable option. The device we envisage for Phase I will incorporate a lateral flow strip as a means of detecting the presence or absence of nucleic acid amplification products by simple visual inspection. BioHelix has experience in developing molecular tests using lateral flow based detection.