Disposable Microfluidic Devices for Point of Care Diagnostics

描述(申请人提供)：虽然大多数急性感染性腹泻是自限性的，但也有一部分需要积极治疗，以最大限度地减少并发症和/或防止传播。一旦出现，几乎不可能知道腹泻病是否会有进行性和/或暴发性病程。因此，提供一种简单、快速、低成本、敏感和特异的诊断测试将使许多急性腹泻的直接治疗成为可能。虽然艰难梭菌相关疾病的检测是这项工作的第一个重点应用，但当这项技术成熟时，也将被应用于广泛的感染性腹泻的床边/现场诊断。事实上，可以在一组可能和/或可能引起腹泻的病原体中识别特定微生物的诊断可能被证明是全球范围内的重要临床流行病学工具，也有望用于生物防御应用(例如，快速识别腹泻疾病的聚集性暴发)。在这份修订的R21申请中，对审查小组提出的问题进行了重大修改，提出了额外的初步数据，并更明确地将拟议的技术定义为适用于一系列疾病监测应用的基础广泛的平台。要测试的假设是，微流控诊断芯片可以在微升规模的粪便样本中检测艰难梭菌，其特异性和敏感性与目前可用的检测相当。这里描述的芯片将能够在几分钟到几小时内完成这些任务，而不是几天。这项工作的目的是：(1)开发一种基于微流控芯片的检测艰难梭菌DNA存在的工具。拟议的工作是采用微流控技术从哺乳动物细胞中分离核酸，并将裂解产物用于细菌样本。样品将在芯片上进行过滤和细胞裂解。分离的核酸将在芯片上使用艰难梭菌特有的引物进行实时聚合酶链式反应分析。(2)确定该系统能否用于检测人类粪便样本中的艰难梭菌。将收集确认感染艰难梭菌的个人的粪便样本，并对其进行鉴定。样品将使用芯片上和芯片外技术进行处理，以比较基于微流控芯片的方法和基于金标准的台式方法。(3)确定基于微流控芯片的诊断方法的特异性和敏感性是否可与最新的酶联免疫吸附试验和艰难梭菌细胞毒素检测相媲美。未知样品的采集、分析和比对将采用微流控芯片和金标准方法。最近在美国和加拿大，艰难梭菌相关感染病例被记录在通常受影响群体之外的患者中：年轻人和不在医院或机构环境中的人。这一进展引起了医学界的极大担忧，因为新的菌株似乎会导致更严重的疾病，所以像这里提出的这样的诊断设备，可以在症状出现时快速区分艰难梭菌和不太严重的感染，对于有效的患者护理至关重要。除了在北美和欧洲使用这种设备治疗新出现的艰难梭菌耐药菌株外，我们还设想(在6-8年内)建立能够区分不同感染性腹泻并指导抗生素供应有限的环境中的治疗的设备。

DESCRIPTION (provided by applicant): While most acute infectious diarrheas are self-limited, a subset requires aggressive treatment in order to minimize complications and/or prevent spread. Upon presentation, it is virtually impossible to know whether a diarrheal illness will have a progressive and/or fulminant course. Thus, the availability of a simple, rapid, low- cost, sensitive and specific diagnostic test would permit the delivery of directed treatment for many acute diarrheas. While testing in C. difficile associated disease is a focused first application of this work, this technology, when mature, will also find application as a bedside/field diagnostic for a wide range of infectious diarrheas. Indeed, a diagnostic that can identify a specific microbe among a panel of likely and/or potential pathogens that cause diarrhea could prove to be an important clinical-epidemiological tool worldwide that also holds promise for biodefense applications (e.g. rapid identification of clustered outbreaks of diarrheal illnesses). In this revised R21 application, significant modifications have been made to address the issues raised by the review panel, to present additional preliminary data, and to more clearly define the proposed technology as a broad-based platform suitable for a range of disease surveillance applications. The hypothesis to be tested is that microfluidic diagnostic chips can detect C. difficile in microliter-scale stool samples with a specificity and sensitivity comparable to presently available testing. The chip described here will be able to perform these tasks in a matter of minutes to hours as opposed to days. The aims of this work are to: (1) Develop a microfluidic chip-based tool to detect the presence of C. difficile bacterial DNA. The proposed work is to adapt a microfluidic technique for isolation of nucleic acids from mammalian cells and lysates to bacterial samples. Samples will be filtered and cells lysed on-chip. The isolated nucleic acids will be analyzed on-chip with real time polymerase chain reaction using primers specific for the C. difficile organism. (2) Determine whether the system can be used to detect C. difficile in human stool samples. Stool samples from individuals confirmed to be infected with C. difficile will be collected and deidentified. Samples will be processed using both on- and off-chip techniques to compare the microfluidic chip-based and gold standard benchtop methods. (3) Determine whether the microfluidic chip based diagnostic is comparable in the specificity and sensitivity to the state of the art ELISA and C. difficile cytotoxin assays. Unknown samples will be collected, analyzed and compared using the microfluidic chip and the gold standard method. Recently in the US and Canada, cases of the C. difficile associated infection has been documented in patients outside of the usual affected groups: younger people and people not in a hospital or institutional environment. This development has been a great cause of concern in the medical community, as the new strains appear to cause a more severe disease, so a diagnostic device, like the one proposed here, that can distinguish C. difficile from less serious infections quickly at the onset of symptoms will be critical for effective patient care. In addition to the use of this device in North America and Europe for emerging resistant strains of C. difficile, we envision building devices (in 6-8 years) that could distinguish between different infectious diarrheas and guide treatment in settings where antibiotic supplies are limited.