Highly Multiplexed Single Molecule Tethering

高度多重单分子系链 总结 有脓毒症症状的患者的血流感染应及时治疗， 抗生素;然而，使用基于血液的方法鉴定病原体所需的时间 培养是及时有效的抗微生物治疗的主要障碍。的发展 一种能够直接从全血中鉴定微生物的综合分子测定法， 由于血液中微生物浓度低， 以及导致血液感染的大量微生物物种（200+）。我们有 开发了一种自动化检测方法，可直接在全血中检测13种血流微生物， 在临床样本中发现的低浓度，LOD在0.5和3 CFU/mL之间， 周转时间70分钟。该检测试剂盒的性能上级于其他FDA批准的检测试剂盒 用于血流病原体检测新检测方法基于单分子连接 （SMOLT），一种新型的样品制备和免扩增分子检测技术 在Scanogen开发。在这里，我们建议大幅增加的复用能力， SMOLT使用一种新的方法，目的是产生一个涵盖生物体的面板 造成超过99%的血流感染病例（目标1）。我们还将整合 将自动化检测试剂盒放入一个一次性检测盒中， 全自动台式仪器（Aim 2）。我们将在初步临床研究中评估新的检测方法 我们将直接比较新检测试剂盒与标准治疗试剂盒的性能 基于血培养的诊断测试，然后进行质谱分析（目标3）。这一切成功都 具体目标有量化的里程碑。 我们的长期目标是开发一个FDA批准和CLIA豁免的系统;该项目 是实现这一目标的关键一步。我们将应对实现所需水平的挑战 多路复用和分析集成，这是开发过程中最严重的风险， 临床系统我们将与一个多学科团队合作，其中包括分析领域的专家。 开发和仪器开发，包括前工程副总裁， 来自约翰霍普金斯的微生物学、脓毒症诊断和治疗专家Becton Dickinson 大学和匹兹堡大学，以及关键技术的工程师。后 完成该项目后，我们将进一步优化系统的可用性和可制造性， 进行分析和临床研究，以提交给FDA。如果成功，新 自动化分析可以成为诊断患者的标准程序的一部分 疑似败血症新的高度多重检测将在其他应用中有用 其中需要快速、成本有效和高度多重的分子检测。

Highly Multiplexed Single Molecule Tethering Summary Bloodstream infections in patients with sepsis symptoms should be promptly treated with antibiotics; however, the time required to identify pathogens using methods based on blood culture is a major obstacle to timely effective antimicrobial therapy. The development of a comprehensive molecular assay capable of identifying microbes directly from whole blood has been elusive despite major efforts due to the low concentration of microbes in the bloodstream and the large number of microbial species that cause bloodstream infection (200+). We have developed an automated assay that detects 13 bloodstream microbes directly in whole blood at the low concentrations found in clinical samples, with LODs between 0.5 and 3 CFU/mL and a turnaround time of 70 minutes. The assay performance is superior to other FDA cleared assays for bloodstream pathogen detection. The new assay is based on Single MOLecule Tethering (SMOLT), a novel sample preparation and amplification-free molecular detection technology developed at Scanogen. Here, we propose to dramatically increase the multiplexing capacity of SMOLT using a novel approach with the goal of producing a panel that covers the organisms responsible for more than 99% of bloodstream infection cases (Aim 1). We will also integrate the automated assay into a single disposable cartridge that will be processed by an easy-to-use and fully automated desktop instrument (Aim 2). We will evaluate the new assay in pilot clinical studies where we will directly compare the performance of the new assay to the standard of care diagnostic test based on blood culture followed by mass spectroscopy (Aim 3). Each of these specific aims has quantitative milestones. Our long-term goal is the development of an FDA-cleared and CLIA-waived system; this project is a critical step towards that goal. We will address the challenges of achieving the required level multiplexing and assay integration, which are the most serious risks on the path to developing the clinical system. We will work with a multidisciplinary team that includes experts in assay development and instrument development, including the former Vice President of Engineering at Becton Dickinson, experts in microbiology, sepsis diagnosis and treatment from Johns Hopkins University and the University of Pittsburgh, as well as engineers from Key Technologies. After completing this project, we will further optimize the system for usability and manufacturability and conduct analytical and clinical studies for submission to the FDA. If successful, the new automated assay can become part of the standard procedure for the diagnosis of patients suspected of having sepsis. The new highly multiplexed assay will be useful in other applications where rapid, cost-effective, and highly multiplexed molecular detection is needed.