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

基本信息

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
项目类别：
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

项目摘要

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，可引起多种局部和全身疾病。面对选择压力，无论是在医院还是在社区环境中，它都会有效地进化出耐药菌株和具有多种毒力因子的菌株。拟议工作的长期目标是开发紧凑型实时 PCR 芯片并将其商业化，以从临床样本中检测金黄色葡萄球菌并对其进行基因分析。原型芯片将有盖玻片大小，并将通过一系列自报告探针进行衍生。由于该阵列将针对每个扩增子上的多个目标区域使用多个探针，因此将实现极高的准确性，并且能够定位每个扩增子内的突变和缺失。该芯片的未来版本将有多个孔，每个孔都涂有自报告探针阵列。在各种潜在的表现形式中，该芯片将提供一个多功能组件，用于集成到各种诊断平台中。该第一阶段应用的具体目标是： 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，我们通常会在与目标相互作用时获得表面结合 BiMB 的 10-100 倍荧光增强。我们将优化我们的表面化学，以促进在片上 RT-PCR 所需的整个温度范围内监测 BiMB。 2) 开发一种热控芯片，使用自我报告“是/否”开关来监测目标抗生素耐药性和毒素基因。该芯片将包括一个与含有目标序列的液相接触的表面相自报告阵列，该阵列被限制在一个热控制室中，该室将通过荧光显微镜实时成像。 3）开发原型片上PCR平台，在RT-PCR过程中监测目标序列扩增，从而识别金黄色葡萄球菌，并确定其是否具有甲氧西林抗性以及是否携带TSST-1和PVL基因。 RT-PCR 芯片原型将设计有多个固定 BiMB 开关，针对每个目标基因的不同区域。芯片结果将根据已在临床环境中得到充分验证的表型和基于溶液的 PCR 测试进行评估。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前开发，该芯片使用微升体积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在于，所提出的诊断装置将提供一种廉价、稳健且精确的方法，用于快速诊断和分析金黄色葡萄球菌感染的抗生素耐药性和各种毒力因子。目前的金黄色葡萄球菌诊断方法通常需要 24 至 48 小时进行初步表征，而所提出的方法将不需要超过两个小时，并将提供无与伦比的诊断准确性的全面表征。