Modified DNA Aptamers and DNAzymes for Diagnosing TB in Resource-Poor Settings

用于在资源匮乏地区诊断结核病的修饰 DNA 适体和脱氧核酶

• 批准号: 8320497
• 负责人: DAN L. FELDHEIM
• 金额: $ 23.01万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8320497
• 关键词: Affinity; Africa; Antibodies; Antigens; Aptamer Technology; Binding; Biological Assay; Biological Markers; Catalytic DNA; Cause of Death; Chemistry; Child; Chimera organism; Clinical; Colorado; Communicable Diseases; Contracts; DNA; DNA Sequence; Detection; Development; Diagnosis; Diagnostic; Diagnostic Procedure; Disease; Electrodes; Enzymes; Equipment; Goals; Gold; HIV Seropositivity; Immunocompromised Host; Infection; Ions; Laboratories; Marketing; Measures; Mediating; Metals; Methods; Mycobacterium tuberculosis; Patients; Plasma; Population; Proteins; RNA Sequences; Reagent; Recording of previous events; Refrigeration; Reporting; Research; Resources; Sampling; Serum; Simulate; Solutions; Southeastern Asia; Specificity; Sputum; Surface; Technology; Testing; Time; Tuberculosis; Universities; Urine; Work; World Health Organization; aptamer; base; cost; instrumentation; lipoarabinomannan; metal complex; nanoparticle; novel diagnostics; operation; professor

DESCRIPTION (provided by applicant): It has been estimated that one-third of the global population (total of ~2 billion) is infected with Mycobacterium tuberculosis (TB). A majority of those infected (90%) will never contract active TB and will remain asymptomatic. In immunocompromised patients, however, TB is a major cause of death worldwide. For example, TB is the leading cause of death in HIV-positive patients. The World Health Organization estimates that 9 million people contracted active TB in 2006 (~6 million from Southeast Asia and Africa alone) and over 1 million died from the disease. Another 10 million are projected to acquire the disease this year, the most in history. Many TB diagnostic assays are on the market or in development. A majority, however, are not well suited for operation in resource-limited settings. Collectively they are too expensive, too time consuming (24 hrs or more), require specialized equipment, expertise, and power, have low sensitivities (50% in the sputum smear test), or do not work on HIV-positive patients or children (antigen tests). It has been estimated that more rapid and accurate TB diagnostic methods would save 400,000 lives per year. The long-term goal of this research is to develop new infectious disease diagnostic assays that can be deployed in resource-limited settings. We seek to develop assays that can be performed outside of a laboratory setting, that is, without PCR amplification, and with minimal power, instrumentation, and scientific expertise. We propose to accomplish this goal by combining two technologies developed at CU-Boulder-modified DNA aptamers and "materials DNAzymes"-with a conductance-based chip platform. Modified DNA aptamers have proven to have affinities and specificities for protein targets that are as good as or better than antibodies. Compared to antibodies, however, DNA aptamers require less time and cost to discover and produce, and are more thermally stable. Materials DNAzymes are DNA sequences that convert otherwise stable metal complex precursors into metal nanoparticles. This application describes the use of modified DNA aptamer/materials DNAzyme conjugates (materials aptazymes) for the electrical detection of TB lipoarabinomannan (LAM) from urine. The Specific Aims for the project are as follows. Aim 1 (Months 1-6). Isolate modified DNA aptamers for TB LAM. Aim 2 (1-6 months). Isolate materials DNAzymes that mediate the formation of Au nanoparticles from solutions containing [Au(Cl)4]1-. Aim 3 (Months 6-12). Synthesize a chimera containing a TB DNA aptamer and Au DNAzyme and verify that each sequence functions properly. Aim 4 (Months 12-24). Determine detection limits and specificities of TB LAM in simulated urine. Upon completion of these aims, we will be poised to use the reagents and technology developed in this project in an RO1 project that will validate LAM in urine as a biomarker of TB infection. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a new diagnostic platform for the detection of infectious disease, with an initial focus on M. tuberculosis (TB). Th technology is intended for use in resource-limited settings, meaning that it will be rapid, portable, and will require little power or significant clinical/scientific expertise to operate.

描述（由申请人提供）：据估计，全球三分之一的人口（总计约20亿）感染结核分枝杆菌（TB）。大多数感染者（90%）永远不会感染活动性结核病，并将保持无症状。然而，在免疫功能低下的患者中，结核病是全世界死亡的主要原因。例如，结核病是艾滋病毒阳性患者死亡的主要原因。世界卫生组织估计，2006年有900万人感染活动性结核病（仅东南亚和非洲就有约600万人），100多万人死于这种疾病。预计今年将有1000万人感染这种疾病，这是历史上最多的。许多结核病诊断测定法已上市或正在开发中。然而，大多数都不太适合在资源有限的环境中运作。总的来说，它们太昂贵，太耗时（24小时或更长时间），需要专门的设备，专业知识和动力，灵敏度低（痰涂片检测为50%），或者不适用于HIV阳性患者或儿童（抗原检测）。据估计，更快速和准确的结核病诊断方法每年可挽救40万人的生命。这项研究的长期目标是开发新的传染病诊断检测方法，可以在资源有限的环境中部署。我们寻求开发可以在实验室环境之外进行的检测方法，即无需PCR扩增，并且只需最少的功率、仪器和科学专业知识。我们建议通过将CU-Boulder开发的两种技术-修饰的DNA适体和“材料DNAzymes”-与基于电导的芯片平台相结合来实现这一目标。经修饰的DNA适体已被证明对蛋白质靶标具有与抗体一样好或更好的亲和力和特异性。然而，与抗体相比，DNA适体需要更少的时间和成本来发现和生产，并且更热稳定。DNA酶是将原本稳定的金属络合物前体转化为金属纳米颗粒的DNA序列。本申请描述了修饰的DNA适体/材料DNA酶缀合物（材料适体酶）用于电检测来自尿的TB脂阿拉伯甘露聚糖（LAM）的用途。该项目的具体目标如下。目标1（1-6个月）。分离用于TB LAM的修饰的DNA适体。目标2（1-6个月）。从含有[Au（Cl）4]1-的溶液中分离介导Au纳米颗粒形成的材料DNA酶。目标3（6-12个月）。合成含有TB DNA适体和Au DNAzyme的嵌合体，并验证每个序列是否正确发挥功能。目标4（12-24个月）。确定模拟尿液中TB LAM的检测限和特异性。在完成这些目标后，我们将准备在RO 1项目中使用该项目中开发的试剂和技术，该项目将验证尿液中的LAM作为结核病感染的生物标志物。 公共卫生关系：该项目的目标是开发一种新的诊断平台，用于检测传染病，最初的重点是M。结核病（TB）。该技术旨在用于资源有限的环境，这意味着它将是快速的，便携式的，并且需要很少的功率或重要的临床/科学专业知识来操作。