Point-of-care microdevices for simultaneous detection of multiple pertussis-like respiratory diseases from Bordetella species

摘要 百日咳，又称百日咳，由B引起。百日咳和B.副百日咳，是 唯一一种疫苗可预防的感染，在美国仍然流行，因为百日咳疫苗- 诱导免疫不是永久性的，免疫宿主在以后的生活中容易再次感染。 全世界估计有2410万百日咳病例，每10年约有160,700人死亡。 这些儿童中大多数来自发展中国家。这两个博德特氏菌 物种具有高度传染性，并导致类似的临床症状，使其难以 诊断。虽然B.副百日咳感染表现出比B更轻的症状。百日咳- 衍生感染，这两种感染都可能是致命的，特别是对儿童，老年人， 免疫功能低下的患者。目前的诊断方法要么速度慢，要么需要昂贵的 仪器.低成本的床旁（POC）检测这两个百日咳引起的 为了满足百日咳快速诊断的需求， 在资源有限的环境中。在我们最近的初步研究中，我们首次开发了 与环介导等温DNA扩增集成的低成本微流控技术 （LAMP）用于B的快速POC检测。百日咳。在此，本提案的目标是发展 一种新的低成本纸/聚合物混合微流体POC装置， 特异性和敏感性诊断两种主要的引起百日咳的博德特氏菌属物种（B. 百日咳和B.副百日咳），其可用于各种场所如医生办公室， 学校和其他低资源环境。我们的核心假设是低- 具有特定DNA测试分析的成本便携式纸基微流体技术可以提供 在资源贫乏的环境中快速、准确和快速的诊断（美国专利#10，875，024）。到 为了实现我们的目标，我们制定了三个具体目标：（1）开发和优化LAMP 用于同时检测B的测定。百日咳和B.（2）开发低成本的 纸/聚合物混合微流体POC装置，用于同时无仪器检测 B。百日咳和B.（3）验证POC设备用于快速检测博德特氏菌 使用临床样本的物种。拟议的方法将允许确认 疑似百日咳病例由不同的博德特氏菌属引起， 填补了目前百日咳诊断方法的空白。此外，我们的生物芯片 快速检测各种其他呼吸道传染病的潜力， 在资源匮乏的环境中应对COVID-19。

ABSTRACT Pertussis, also known as whooping cough, caused by B. pertussis and B. parapertussis, is the only vaccine-preventable infection that remains endemic in the U.S. Since pertussis vaccine- induced immunity is not permanent, the immunized host is vulnerable to re-infection later in life. Worldwide, there are an estimated 24.1 million cases of pertussis, and about 160,700 deaths per year from pertussis in children, most of which are from developing nations. These two Bordetella species are highly contagious and cause similar clinical symptoms, making them difficult to diagnose. Although B. parapertussis infection manifests in milder symptoms to B. pertussis- derived infection, both infections can be deadly, especially to children, the elderly, and immunocompromised patients. Current diagnostic methods are either slow or require costly instruments. Low-cost point-of-care (POC) detection of these two whooping cough-causing pathogens is greatly needed to meet the unmet demand for rapid diagnosis of whooping cough in resource-limited settings. During our recent preliminary study, we, for the first time, developed a low-cost microfluidic technology integrated with the loop-mediated isothermal DNA amplification (LAMP) for rapid POC detection of B. pertussis. Herein, the goal of this proposal is to develop a new low-cost paper/polymer hybrid microfluidic POC device integrated with LAMP for rapid, specific, and sensitive diagnosis of two major whooping cough-causing Bordetella species (B. pertussis and B. parapertussis) that can be used in various venues such as physicians’ offices, schools, and other low-resource settings. Our central hypothesis is that the integration of a low- cost portable paper-based microfluidic technology with specific DNA testing assays can provide a fast, accurate, and rapid diagnosis in resource-poor settings (US patent # 10,875,024). To accomplish our goal, we have developed three specific aims: (1) Develop and optimize LAMP assays for simultaneous detection of B. pertussis and B. parapertussis; (2) Develop a low-cost paper/polymer hybrid microfluidic POC device for the simultaneous instrument-free detection of B. pertussis and B. parapertussis; and (3) Verify the POC device for rapid detection of Bordetella species using clinical samples. The proposed methodology will allow for the confirmation of suspected cases of whooping cough caused by different Bordetella species at the point of care, filling a gap of current diagnostic methods in whooping cough. Furthermore, our biochip has great potential for the rapid detection of a variety of other respiratory infectious diseases such as COVID-19 in resource-poor settings.