A Culture-Free Platform for Rapid Pathogen Identification

描述（由申请方提供）：用于快速病原体鉴定的无培养平台项目总结脓毒症是美国发病率和死亡率的主要原因之一，每年发生超过751，000例确诊病例，210，000例死亡。这种高死亡率（约30%）意味着每天有近600名患者死于败血症相关感染。除了人员伤亡，经济负担也同样巨大，在美国达到240亿美元/年，平均每位患者的费用超过23，000美元。正确治疗脓毒症的关键是快速准确地鉴定致病微生物（细菌或真菌）。不幸的是，鉴于可能诱发败血症的潜在病原体范围广泛，当今诊断的“金标准”仍然是培养;这通常需要长达3-5天的时间才能准确识别，导致治疗显着延迟 启动靶向治疗方案。该小型企业创新研究I期项目的目的是证明一种新型无培养测定法的可行性，该测定法用于在几小时内检测和鉴定临床上最普遍的脓毒症诱导真菌病原体。该项目中提出的创新消除了对耗时和不敏感培养的需要，从而将从标本到结果的时间从几天显著缩短到几个小时。此外，由于不需要物种特异性扩增，我们的方法 更快，并且直接适合于多路复用。我们的新工艺利用了一种新的合成核酸类似物γ-PNA，其具有通过标准沃森-克里克碱基配对规则以无与伦比的序列特异性侵入dsDNA的能力。我们在此应用中的重点是在第一阶段项目范围内实现特定的概念验证。本阶段I的性能目标是建立一种采用γ-PNA探针的测定方法，能够识别和区分最突出的脓毒症诱导真菌病原体。此外，在成功证明我们的测定后，我们将滴定测定至10 CFU。我们将通过在样本中加入越来越多的背景人类DNA来完成我们的研究，确定真实世界水平的非靶DNA不会阻碍诊断过程。在实现这些目标后，在第二阶段，我们将开发一个原型系统，直接从静脉切开术样本工作，并解决所有问题，如优化，自动化和商业格式。鉴于我们的方法可以轻松地进行多路复用，未来将努力通过增加我们小组中病原体（细菌和真菌）的数量来扩大我们的检测试剂盒，最终导致“一次检测系统”的开发，用于无培养鉴定约20种最常见的诱导脓毒症反应的微生物病原体。γ-PNA探针是用于病原体鉴定的有吸引力的方法，因为它们提供显著的序列特异性以及极高的靶亲和力，从而促进更灵敏和更特异的读出。所提出的检测方法将使诊断真菌血流感染的方法更加快速，简化和准确，使低成本的方法能够快速有效地降低目前与败血症相关的无法忍受的高死亡率水平。

DESCRIPTION (provided by applicant): A Culture-Free Platform for Rapid Pathogen Identification Project Summary Sepsis is a one of the leading causes of morbidity and mortality in the US, with more than 751,000 confirmed cases occurring every year and 210,000 mortalities. This high mortality rate (~30%) means that nearly 600 patients a day succumb to sepsis-related infections. In addition to the human toll, the economic burden is likewise immense, reaching $24B/year in the US, with an average cost of over $23,000 per patient. The key to treating sepsis properly is rapid and accurate identification of the causative microorganism (bacterial or fungal). Unfortunately, given the wide range of potential pathogens which can induce sepsis, the 'gold-standard' today for diagnosis remains culturing; which typically requires up to 3-5 days for accurate identification leading to a significant delay in the initiation of a targeted treatment protocol. The objective of this Small Business Innovation Research Phase I project is to demonstrate feasibility of a novel, culture- free assay for detecting and identifying the most clinically prevalent sepsis inducing fungal pathogens in a matter of hours. The proposed innovation in this project removes the need for time consuming and insensitive culturing, thus significantly decreasing the time from specimen to result from days to a few hours. Additionally as species-specific amplification is not required, our process is faster and is directly amicable to multiplexing. Our new process utilizes a new synthetic nucleic acid analogue, gamma-PNA, which has the demonstrated ability to invade dsDNA with unmatched sequence specificity through standard Watson-Crick base pairing rules. Our focus in this application is a specific proof-of-concept to be achievable within the scope of a Phase I project. The performance goal of this Phase I is to establish an assay employing gamma-PNA probes, capable of identifying and distinguishing among the most prominent sepsis inducing fungal pathogens. Furthermore, after successfully demonstrating our assay, we will titrate the assay down to 10 CFU. We will culminate our study by spiking the samples with increasing amounts of background human DNA, establishing that real-world levels of non-target DNA do not hinder the diagnostic process. Having achieved these goals, in Phase II we will develop a prototype system, working directly from phlebotomy samples and address all issues such as optimization, automation and commercial format. Given the ease in which our approach can be multiplexed, future efforts will be made to expand the repertoire of our assay by increasing the number of pathogens (to both bacterial and fungal) in our panel ultimately leading to the development of a 'one-assay system' for the culture-free identification of the ~20 most prevalent microbial pathogens which induce a septic response. Gamma-PNA probes are an attractive approach for pathogen identification as they offer remarkable sequence specificity as well as extremely high target affinity facilitating a more sensitive and more specific readout. The proposed assay would enable a dramatically faster, simplified and more accurate method of diagnosing fungal blood stream infections, enabling a low-cost method to rapidly and efficiently reduce the unbearably high mortality levels currently associated with sepsis.