Self-Reporting Arrays for Rapid, Robust and Precise S. aureus Diagnostics

用于快速、稳健和精确金黄色葡萄球菌诊断的自我报告阵列

• 批准号: 7644518
• 负责人: WILLIAM H BRAUNLIN
• 金额: $ 30万
• 依托单位: RATIONAL AFFINITY DEVICES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7644518
• 关键词: Antibiotic Resistance; Bacteria; Binding; Biological Assay; Blood; Cerebrospinal Fluid; Chemistry; Clinical; Complement; Complex; Correlation Studies; Deletion Mutation; Development; Devices; Diagnosis; Diagnostic; Diagnostic Procedure; Drug resistance; Enterococcus faecalis; Face; Fluorescence; Fluorescence Microscopy; Future; Gene Targeting; Genes; Genetic; Goals; Gold; Hospitals; Hour; Image; Infection; Liquid substance; Measurement; Methicillin Resistance; Methods; Mind; Modification; Molecular; Monitor; Nature; Nose; Panton-Valentine leukocidin; Patient Self-Report; Performance; Phase; Problem Solving; Property; Reproducibility; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Sensitivity and Specificity; Series; Solutions; Sputum; Staphylococcus aureus; Surface; Swab; System; Systemic disease; Temperature; Testing; Time; Toxic Shock Syndrome Toxin-1; Toxin; Urine; Vancomycin Resistance; Virulence Factors; Work; base; clinically relevant; community setting; cost; design; diagnostic accuracy; meetings; melting; pathogen; pressure; prototype; public health relevance; research study; resistant strain

DESCRIPTION (provided by applicant): Staphylococcus aureus is a highly successful bacterium that causes a wide range of local and systemic diseases. In the face of selective pressure, it evolves efficiently to develop drug-resistant strains and strains with a variety of virulence factors, both in hospital and in community settings. The long-term goal of the proposed work is to develop and commercialize a compact real-time PCR chip to detect and genetically profile S. aureus from clinical samples. The prototype chip will be the size of a cover-slip, and will be derivatized with an array of self-reporting probes. Because this array will utilize multiple probes for multiple target regions on each amplicon, an exceptionally high level of accuracy will be achieved, along with the ability to localize mutations and deletions within each amplicon. Future versions of the chip will have multiple wells, each coated with arrays of self-reporting probes. In a variety of potential manifestations, the chip will provide a versatile component for integration into a wide range of diagnostic platforms. The specific aims of this Phase I application are: 1) To optimize surfaces for the functional attachment of bimolecular beacons (BiMBs) for variable temperature applications. We now routinely obtain 10-100 fold fluorescence enhancements of surface-bound BiMBs upon interaction with target. We will optimize our surface chemistries to facilitate monitoring of BiMBs over the full range of temperatures necessary for on-chip RT-PCR. 2) To develop a thermally controlled chip that uses self-reporting YES/NO switches in order to monitor target antibiotic resistance and toxin genes. The chip will comprise a surface-phase self-reporting array in contact with a liquid phase containing target sequences, confined within a thermally controlled chamber that will be imaged in real-time by fluorescence microscopy. 3) To develop a prototype on-chip PCR platform that monitors target sequence amplification during the course of RT-PCR, and thereby functions to identify S. aureus, and to determine if it is methicillin resistant, and whether it harbors the TSST-1 and PVL genes. The prototype RT- PCR chip will be designed with multiple fixed BiMB switches directed against different regions for each target gene. The chip results will be evaluated against phenotypic and solution-based PCR tests that have been well- validated in clinical settings. In phase II of this project, the focus will be on pre-commercial development of rapid, sensitive, specific and reliable S. aureus diagnostic chips that use microliter volumes of a variety of clinically relevant sample types. PUBLIC HEALTH RELEVANCE The public health relevance of this work is that the proposed diagnostic device will provide an inexpensive, robust, and precise means of rapidly diagnosing and profiling S. aureus infections with respect to antibiotic resistance and to a variety of virulence factors. Whereas current S. aureus diagnostic methods generally take from 24 to 48 hours for a preliminary characterization, the proposed method will take no more that two hours, and will provide a comprehensive characterization of unparalleled diagnostic accuracy.

描述(申请人提供)：金黄色葡萄球菌是一种非常成功的细菌，可引起广泛的局部和全身疾病。面对选择的压力，在医院和社区环境下，高效地发展耐药菌株和具有多种毒力因子的菌株。这项拟议工作的长期目标是开发一种紧凑的实时聚合酶链式反应芯片并将其商业化，以检测临床样本中的金黄色葡萄球菌并对其进行基因分析。原型芯片将是盖子大小的芯片，并将通过一系列自我报告的探头进行衍生。由于该阵列将针对每个扩增子上的多个靶区使用多个探针，因此将实现异常高的准确度，以及定位每个扩增子内的突变和缺失的能力。未来版本的芯片将有多个井，每个井都涂有自报告探头阵列。在各种潜在的表现中，该芯片将提供一个多功能组件，用于集成到广泛的诊断平台中。这一第一阶段应用的具体目标是：1)为变温应用中双分子信标(BiMBs)的功能附着优化表面。我们现在常规地获得10-100倍的表面结合的BiMBs与靶相互作用的荧光增强。我们将优化我们的表面化学性质，以便于在芯片上RT-PCR所需的全温度范围内监测BiMBs。2)开发一种热控芯片，该芯片使用自我报告的是/否开关，以监测目标抗生素耐药性和毒素基因。该芯片将包括一个表面相自报告阵列，与含有靶标序列的液体接触，被限制在一个将通过荧光显微镜实时成像的热控室中。3)建立一个芯片上的原型聚合酶链式反应平台，在RT-PCR过程中监测目标序列的扩增，从而鉴定金黄色葡萄球菌，确定它是否对甲氧西林耐药，以及它是否含有TSST-1和PVL基因。原型RT-PCR芯片将设计有多个固定的BIMB开关，针对每个目标基因的不同区域。芯片结果将根据表型和基于溶液的PCR测试进行评估，这些测试已经在临床环境中得到了很好的验证。在该项目的第二阶段，重点将是快速、灵敏、特异和可靠的金黄色葡萄球菌诊断芯片的商业化前开发，这些芯片使用微升容量的各种临床相关样本类型。公共卫生相关性这项工作的公共卫生相关性是，拟议的诊断设备将提供一种廉价、强大和准确的手段，以快速诊断和描述关于抗生素耐药性和各种毒力因素的金黄色葡萄球菌感染。目前的金黄色葡萄球菌诊断方法一般需要24到48小时进行初步鉴定，而建议的方法不会超过两个小时，并将提供无与伦比的诊断准确性的全面表征。