Rapid, Handheld, Point-of-Care Stool Sample-prep Device for the Extraction and Detection of Nucleic Acid from Diarrheal Pathogens

摘要 腹泻病是世界范围内的一个主要健康问题，每年导致近50万人死亡 适用于5岁以下儿童。那些幸存下来的人发育迟缓的风险更高， 认知发展。引起腹泻的胃肠道(GI)病原体的鉴定 疾病有助于指导有效的治疗。目前，在发达国家，这是通过使用 多重聚合酶链式反应实验室检测昂贵、缓慢，并需要复杂的高 复杂性实验室。不幸的是，这样的实验室测试不能在偏远和 发展中世界资源有限的地区，腹泻死亡率最高。 因此，我们建议开发一种集成的、低成本的护理点(POC)测试来识别GI 引起腹泻疾病的病原体。此测试在以下资源受限的设置中非常有用 发展中世界以及发达国家的初级保健和紧急护理环境 在临床医生的办公室里，实时检测胃肠道病原体是有用的，所以 患者可以立即用正确的药物治疗，而不会失去患者的随访。这个 检测胃肠道(GI)病原体的主要问题是粪便样本的复杂性， 通常充满微粒和分子，如胆盐。这种污染物可以抑制 信号放大和检测，如果没有从样品中适当去除。在以前的SBIR中 项目，GoDx开发了样品制备化学、DNA扩增和检测 一组胃肠道病原体的侧向流动试纸检测。我们的下一步是开发一种 完全集样品处理和信号放大于一体的病原体检测装置， 其可以在护理点(PoC)快速使用。为了实现这一目标，我们建议制定一项 一种可为扩增和检测ALL提供活DNA的粪便标本制备装置 同时需要最小的用户动作来这样做。这里提出的技术将能够 在几分钟内从粪便拭子中提取和丰富DNA，而不需要 离心机或泵。在这个项目的第二阶段，我们将完全整合GoDx的放大 而检测分析与这里开发的样品制备技术，提供了一种无缝的， 用于检测引起腹泻疾病的胃肠道病原体的最小步长POC装置 环游世界。

Abstract Diarrheal disease is a major health issue worldwide resulting in nearly 500,000 deaths annually in children under 5 years of age. Those who survive have higher risk of stunted growth and cognitive development. Identification of the gastrointestinal (GI) pathogens that cause diarrheal disease helps guide effective treatment. Currently, in the developed world, this is done using multiplexed PCR laboratory tests which are expensive, slow and require a sophisticated high complexity laboratory. Unfortunately, such laboratory tests are not available in remote and resource limited regions in the developing world where mortality due to diarrhea is the highest. Thus, we propose to develop an integrated, low-cost, point-of-care (POC) test to identify the GI pathogens that cause diarrheal diseases. This test will be useful in resource-limited settings of the developing world as well as in primary care and urgent care settings of the developed world where it will be useful to detect the GI pathogens in real time, in the clinician's office so the patient can be treated with the right drugs, right away, without loss of patients to follow up. The major problem with detecting gastrointestinal (GI) pathogens is the complexity of stool samples, often filled with particulate and molecules such as bile salts. Such contaminants can inhibit signal amplification and detection if not properly removed from the sample. In previous SBIR projects, GoDx has developed sample preparation chemistry, DNA amplification and detection assays on lateral flow paper strips for a suite of GI pathogens. Our next step is to develop a pathogen detection device that fully integrates sample processing and signal amplification, which can be used rapidly at the point of care (POC). To achieve this, we propose to develop a stool sample prep device that can robustly provide viable DNA for amplification and detection all while requiring minimal user action to do so. The technology proposed here will be able to extract and enrich DNA from a swab of stool in a matter of minutes without the need for centrifuges or pumps. In Phase 2 of this project, we will fully integrate the GoDx’s amplification and detection assays with the sample prep technology developed here, providing a seamless, minimal step POC device for the detection of GI pathogens that cause diarrheal diseases around the world.