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

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，可引起广泛的局部和全身性疾病。面对选择性压力，无论是在医院还是在社区环境中，它都能有效地发展出耐药菌株和具有各种毒力因子的菌株。这项工作的长期目标是开发和商业化一种紧凑的实时PCR芯片来检测和遗传分析S。金黄色葡萄球菌。原型芯片将是一个盖玻片的大小，并将与一个阵列的自我报告探针衍生。因为该阵列将利用多个探针用于每个扩增子上的多个靶区域，所以将实现异常高水平的准确性，沿着定位每个扩增子内的突变和缺失的能力。未来的芯片将有多个威尔斯孔，每个孔都涂有自我报告探针阵列。在各种潜在的表现形式中，该芯片将提供一个多功能组件，用于集成到广泛的诊断平台中。该阶段I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）的功能性附着的表面。我们现在常规地获得与靶相互作用后表面结合的BiMB的10-100倍荧光增强。我们将优化我们的表面化学，以促进在芯片上RT-PCR所需的整个温度范围内监测BiMB。2)开发一种使用自我报告YES/NO开关的热控芯片，以监测目标抗生素耐药性和毒素基因。该芯片将包括与含有靶序列的液相接触的表面相自报告阵列，该阵列被限制在热控制室内，该热控制室内将通过荧光显微镜实时成像。3)建立一个芯片上PCR原型平台，在RT-PCR过程中监测靶序列扩增，从而实现对沙门氏菌的鉴定。金黄色葡萄球菌，并确定它是否是耐甲氧西林的，以及它是否携带TSST-1和PVL基因。原型RT-PCR芯片将设计有针对每个靶基因的不同区域的多个固定BiMB开关。将根据已在临床环境中充分验证的表型和基于溶液的PCR检测来评价芯片结果。在该项目的第二阶段，重点将是快速、灵敏、特异和可靠的S。使用微升体积的各种临床相关样品类型的金黄色葡萄球菌诊断芯片。公共卫生相关性这项工作的公共卫生相关性是，所提出的诊断设备将提供一种快速诊断和分析S的廉价、稳健和精确的方法。金黄色葡萄球菌感染的抗生素耐药性和各种毒力因子。而目前的S.金黄色葡萄球菌的诊断方法通常需要24至48小时进行初步表征，而本发明的方法将不需要超过两小时，并且将提供无与伦比的诊断准确性的全面表征。

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.