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。面对选择压力，它在医院和社区环境中都有效地进化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一种紧凑的实时PCR芯片并将其商业化，以检测临床样品中的金黄色葡萄球菌并对其进行遗传分析。原型芯片将是一个盖片大小，并将衍生出一系列自我报告探针。由于该阵列将对每个扩增子上的多个目标区域使用多个探针，因此将实现非常高的准确性，以及在每个扩增子内定位突变和缺失的能力。未来版本的芯片将有多个孔，每个孔上都涂有自我报告探针阵列。在各种潜在表现中，该芯片将提供一个通用组件，用于集成到广泛的诊断平台中。第一阶段应用的具体目标是：1)优化可用于可变温度应用的双分子信标（bimb）的功能附着表面。我们现在通常在与目标相互作用时获得10-100倍的表面结合BiMBs荧光增强。我们将优化我们的表面化学，以方便在片上RT-PCR所需的全温度范围内监测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。该芯片将包括一个表面相自报告阵列，该阵列与含有目标序列的液相接触，被限制在一个热控室中，将通过荧光显微镜实时成像。3)开发芯片上PCR平台原型，在RT-PCR过程中监测靶序列扩增，从而鉴定金黄色葡萄球菌，判断其是否耐甲氧西林，是否携带TSST-1和PVL基因。原型RT- PCR芯片将设计多个固定的BiMB开关，针对每个靶基因的不同区域。芯片结果将对表型和基于溶液的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，重点将放在快速、敏感、特异和可靠的金黄色葡萄球菌诊断芯片的预商业化开发上，该芯片使用微升体积的各种临床相关样品类型。这项工作的公共卫生意义在于，提出的诊断设备将提供一种廉价、可靠和精确的方法，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素。目前的金黄色葡萄球菌诊断方法通常需要24至48小时进行初步鉴定，而拟议的方法将不超过2小时，并将提供无与伦比的诊断准确性的全面鉴定。

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.