Integrated molecular diagnostic system for the point-of-care

用于现场护理的综合分子诊断系统

• 批准号: 8056922
• 负责人: Huimin Kong
• 金额: $ 24.19万
• 依托单位: BIOHELIX CORPORATION
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8056922
• 关键词: Bedside Testings; Biological Assay; Boston; Buffers; Centers for Disease Control and Prevention (U.S.); Chemistry; Chlamydia trachomatis; Client; Clinic; Clinical; Cytolysis; DNA; DNA amplification; Detection; Device Designs; Devices; Diagnostic; Engineering; Eye; Hand; Health Personnel; Housing; Laboratories; Lateral; Learning; Liquid substance; Literature; Manufacturer Name; Marketing; Microfluidic Microchips; Molecular; Movement; National Institute of Allergy and Infectious Disease; Neisseria gonorrhoeae; Nucleic Acids; Patients; Phase; Point-of-Care Systems; Polymerase Chain Reaction; Process; Reaction; Reader; Reagent; Research; Risk; Sampling; Sexually Transmitted Diseases; Small Business Technology Transfer Research; Solid; Solutions; Staging; Surgical Flaps; System; Technical Expertise; Technology; Temperature; Test Result; Testing; Tube; Universities; Urine; Validation; Vent; Visual; Work; base; commercialization; cost; design; experience; fluorescence microscope; helicase; high throughput screening; inhibitor/antagonist; instrument; internal control; micro-total analysis system; pathogen; point of care; prototype

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.

描述（由申请人提供）：这种Sttr-at-Niaid试图根据波士顿大学凯瑟琳·克拉普里奇（Catherine Klapperich）的实验室进行的研究开发综合的核酸系统。 BU实验室芯片包括微型固相提取（<spe）柱，瓣阀和疏水通风孔通过微通道，以及用于实验重复和控制反应的多个反应室。 Klapperich博士的实验室已使用Biohelix的等温扩增过程在芯片中成功地进行了核酸扩增。 Biohelix的专有技术称为解旋酶依赖性扩增（HDA）。它在指数放大过程中使用DNA解旋酶在65：c的恒定温度下分离DNA链。像聚合酶链反应（PCR）一样，HDA测定使用竞争性的内部对照（即，已知浓度的模板DNA中飙升到原始样品中的模板DNA）可以通过与分析物相同的引物进行扩增，但被分别检测到。因此，我们可以在直接临床样品中检测放大抑制剂。 第一阶段的目标是：1）修改BU微流体设备设计以结合横向流条作为使用肉眼检测放大产品的手段； 2）证明使用AIM 1的设备使用CT进行集成测试的可行性，并使用CT和NG在104 in 1 ml尿液中升高，以建立概念验证验证验证验证，以在包括横向流动条的改良的BU芯片中进行的集成测定法。我们对第二阶段的具体目的是开发横向流量读取器子系统，将其与BU仪器集成在一起以构建像商业前外观一样 /类似于工作的原型，简化芯片设计和制造，以便进行飞行量表制造，并在Biohelix / Quidel上为一次性的飞行员制造。在此阶段，Qiagen是读者最有可能的制造商。在第二阶段结束时，我们应该准备好对新测定系统的临床验证。 公共卫生相关性：此I阶段的Sttr-at-Niaid项目寻求概念证明项目将重点关注最丰富的性传播疾病（STD）病原体：沙眼衣原体（CT）和Neisseria Gonorrhoeae（NG）。科学文献清楚地表明，分子测试是检测CT和NG的最敏感方法，而分子CT/NG高吞吐量筛选市场目前的价值超过3亿美元/年。此外，如果医疗保健工作者怀疑这些患者不太可能返回STD诊所学习测试结果，则CDC敦促性病诊所通过POC检测患者进行测试。不幸的是，没有辅助点（POC）CT NG分子测试，而现有的POC分子测试系统（例如GenExpert）对于在STD诊所中使用太高了。这个拟议的项目将寻求纠正这一短暂的到来。 我们建议使用由波士顿大学（BU）的凯瑟琳·克拉普里奇（Catherine Klapperich）博士开发的设计来开发低成本的POC分子诊断系统。尽管当前的BU可支配可以执行我们的专有等温扩增反应，但它不允许低成本，无仪器的扩增产物检测；即，荧光显微镜可用于检测产品形成，但这不是商业上可行的选择。我们设想的第I期设备将结合横向流带，以作为通过简单的视觉检查来检测存在或不存在核酸扩增产物的手段。 Biohelix具有使用基于横向流的检测来开发分子测试的经验。