Ultra-sensitive magnetic assays for quantitative detection of nucleic acids witho

用于定量检测核酸的超灵敏磁性测定

• 批准号: 7746826
• 负责人: Mark S. DiIorio
• 金额: $ 18.52万
• 依托单位: MAGNESENSORS, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7746826
• 关键词: Aliquot; Antibiotic Resistance; Antibodies; Area; Bacteria; Base Sequence; Benchmarking; Binding; Biological Assay; Biomedical Engineering; Blood; Blood specimen; Buffers; Bypass; Cancer Detection; Cell Surface Receptors; Chemistry; Clinical; Clinical Research; Communicable Diseases; Community Hospitals; Contracts; Detection; Development; Devices; Diagnosis; Diagnostic; Enzymes; Escherichia coli; Escherichia coli O157; Figs - dietary; Generations; Genomics; Genus staphylococcus; Goals; High temperature of physical object; Hour; Immunoassay; Infection; Interleukin-6; Label; Laboratories; Lead; Legal patent; Magnetism; Marketing; Measurement; Measures; Messenger RNA; Methods; Microspheres; Molecular; Monitor; Nanotechnology; Nature; Nosocomial Infections; Nucleic Acid Amplification Tests; Nucleic Acid Probes; Nucleic Acids; Nucleic acid sequencing; Organism; Pathogen detection; Penetration; Peptide Nucleic Acids; Phase; Plasma; Polystyrenes; Preparation; Prevention; Principal Investigator; Publications; RNA; Randomized; Reagent; Research; Residual Neoplasm; Resistance; Ribosomal RNA; Sampling; Sensitivity and Specificity; Sepsis; Serum; Signal Transduction; Small Business Innovation Research Grant; Source; Species Specificity; Specificity; Specimen; Speed; Staphylococcus aureus; Streptavidin; System; Techniques; Technology; Testing; Time; Training; United States National Aeronautics and Space Administration; United States National Institutes of Health; Whole Blood; Work; Writing; base; cost; cost effective; design; experience; fiberglass; improved; infectious disease treatment; innovation; instrument; instrumentation; magnetic field; methicillin resistant Staphylococcus aureus; nanoparticle; novel; nucleic acid detection; pathogen; pathogenic bacteria; professor; programs; prototype; public health relevance; rapid detection; sensor; success; superconducting quantum interference device; synthetic nucleic acid

DESCRIPTION (provided by applicant): MagneSensors' program is aimed at developing a rapid, ultra-sensitive magnetic assay to quantitatively detect nucleic acids without the need for PCR or other amplification. The novel sandwich hybridization assay takes advantage of an exquisitely sensitive magnetic sensor that measures the magnetic field from magnetic nanoparticle labels on nucleic acid probes hybridized to RNA targets. There are many applications in clinical diagnostics, such as the detection of pathogens causing sepsis, where amplified nucleic acid tests have struggled to gain acceptance because of drawbacks including false positives and false negatives due to contamination and other issues. Much effort has been expended on direct detection methods but the challenges in achieving high sensitivity on clinical specimens have proven formidable and limited applications. Magnetic detection offers unique advantages such as high sensitivity, specificity, speed, and simplicity, a combination that is very difficult to achieve in competing platforms. Our latest generation bench top instrument uses a high temperature superconducting quantum interference device (SQUID) that is cooled with a small cryorefrigerator and is capable of measuring 50 assay samples in a minute at very high sensitivity. The user needs not even be aware of the cooling, which has been an often-cited drawback for using SQUIDs, and the instrument is well-suited for clinical diagnostics. The Phase I aim is to demonstrate magnetic detection of ribosomal RNA (rRNA) in Staphylococcus aureus with the following benchmarks: 1) 6 x 104 copies rRNA (100 zmol), equivalent to H 20 CFU/ml S. aureus in 1 ml sample volume, 2) specificity of S. aureus vs. S. epidermidis control, 3) 45 minutes assay preparation and measurement (exclusive of RNA extraction) and 4) no separation of unbound magnetic nanoparticle labels. The assays thus take advantage of the abundance of rRNA (500-2,000 copies) in each bacterium. Another innovative aspect is the use of peptide nucleic acid (PNA) probes. These synthetic nucleic acids offer advantages of better binding, increased specificity, higher stability, reduced hybridization time, and resistance to degradation by enzymes. It is expected that the above aims would be much further improved in Phase II, enabling detection down to a single bacterium as well as specific detection of methicillin resistant Staphylococcus aureus (MRSA) in a total assay time of 30-45 minutes. PUBLIC HEALTH RELEVANCE: The ability to quickly and easily perform quantitative measurement of nucleic acids without amplification would open the door to a host of important applications. Development of an ultra-sensitive platform for rapid, accurate, and cost effective bacterial detection systems would revolutionize the diagnosis, prevention, and treatment of infectious diseases. These include community and hospital acquired infections such as staphylococcus resistant to antibiotics (MRSA), which are on the rise and often lead to sepsis.

描述（由申请人提供）：MagneSensors的项目旨在开发一种快速、超灵敏的磁性测定法，以定量检测核酸，而无需PCR或其他扩增。新的夹心杂交检测利用了一种非常灵敏的磁性传感器，该传感器测量来自与RNA靶杂交的核酸探针上的磁性纳米颗粒标签的磁场。在临床诊断中有许多应用，例如检测引起败血症的病原体，其中扩增核酸测试由于包括由于污染和其他问题引起的假阳性和假阴性的缺点而难以获得接受。在直接检测方法上已经花费了很多努力，但是在临床标本上实现高灵敏度的挑战已经证明是强大的且有限的应用。磁性检测具有独特的优势，如高灵敏度，特异性，速度和简单性，这是在竞争平台中很难实现的组合。我们最新一代的台式仪器使用高温超导量子干涉仪（SQUID），该仪器通过小型低温制冷器冷却，能够在一分钟内以极高的灵敏度测量50个分析样品。用户甚至不需要意识到冷却，这是使用SQUID的一个经常提到的缺点，并且该仪器非常适合临床诊断。第一阶段的目标是证明磁性检测金黄色葡萄球菌中的核糖体RNA（rRNA），具有以下基准：1）6 x 104拷贝rRNA（100 zmol），相当于H 20 CFU/ml S。金黄色葡萄球菌的特异性;金黄色葡萄球菌vs.表皮对照，3）45分钟测定制备和测量（不包括RNA提取）和4）未分离的磁性纳米颗粒标记。因此，该测定利用了每种细菌中rRNA的丰度（500- 2，000拷贝）。另一个创新方面是使用肽核酸（PNA）探针。这些合成核酸提供了更好的结合、增加的特异性、更高的稳定性、减少的杂交时间和对酶降解的抗性的优点。预计上述目标将在第II阶段得到进一步改进，使得能够在30-45分钟的总测定时间内检测到单个细菌以及特异性检测耐甲氧西林金黄色葡萄球菌（MRSA）。公共卫生相关性：在没有扩增的情况下快速和容易地进行核酸定量测量的能力将为许多重要应用打开大门。开发用于快速、准确和具有成本效益的细菌检测系统的超灵敏平台将彻底改变传染病的诊断、预防和治疗。这些包括社区和医院获得性感染，如耐药性抗生素（MRSA），这是在上升，往往导致败血症。