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: 7846523
• 负责人: WILLIAM H BRAUNLIN
• 金额: $ 0.63万
• 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 2009-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7846523
• 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（由申请人提供）：金黄色葡萄球菌是一种非常成功的细菌，可引起各种局部疾病和全身性疾病。面对选择性压力，它在医院和社区环境中都具有各种毒力因素的抗药性和菌株有效发展。拟议工作的长期目标是开发和商业化紧凑的实时PCR芯片，以检测并从临床样品中概述金黄色葡萄球菌。原型芯片将是盖玻片的大小，并将用一系列自我报告的探针衍生。因为该数组将对每个扩增子上的多个目标区域使用多个探针，因此将实现异常高的准确性，以及在每个扩增子内定位突变和缺失的能力。芯片的未来版本将有多个井，每个井都带有一系列自我报告的探针。在各种潜在的表现中，芯片将为整合到广泛的诊断平台中提供多功能组件。该阶段应用程序的具体目的是：1）优化用于可变温度应用的双分子信标（BIMB）功能附着的表面。现在，我们通常在与目标相互作用后常规获得表面结合的BIMB的10-100倍荧光增强。我们将优化表面化学，以促进在片上RT-PCR所需的全部温度下监测BIMB的监测。 2）开发使用自我报告的是/否开关的热控制芯片，以监测靶抗生素耐药性和毒素基因。该芯片将包含一个与液相接触的表面自我报告阵列，该阵列包含液相，该阵列包含靶序列，该阵列被限制在热控制室内，该腔室将通过荧光显微镜实时成像。 3）开发一个原型片上PCR平台，该平台在RT-PCR过程中监视靶向序列扩增，从而起作用以识别金黄色葡萄球菌，并确定它是否具有甲氧西林抗性，以及它是否具有TSST-1和PVL基因。原型RT-PCR芯片将使用针对每个目标基因的不同区域的多个固定BIMB开关设计。芯片结果将根据在临床环境中得到良好验证的表型和基于溶液的PCR检验进行评估。在该项目的第二阶段中，重点将放在快速，敏感，特定和可靠的金黄色葡萄球菌诊断芯片的商业前开发上，这些芯片使用各种临床相关样本类型的微量尺寸。公共卫生相关性的这项工作的公共卫生相关性是，拟议的诊断装置将提供廉价，健壮和精确的手段，用于快速诊断和分析金黄色葡萄球菌感染有关抗生素耐药性以及多种毒力因素。当前的金黄色葡萄球菌诊断方法通常需要从24到48小时才能进行初步表征，但提出的方法将不再需要两个小时，并且将提供无与伦比的诊断准确性的全面表征。