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，由百日咳杆菌和百日咳杆菌引起。 自百日咳疫苗接种以来，唯一在美国流行的疫苗可预防的感染- 诱导免疫不是永久性的，免疫的宿主在以后的生命中很容易再次感染。 全世界估计有2410万百日咳病例，每年约有160700人死亡。 从儿童百日咳开始的一年，其中大部分来自发展中国家。这两个波尔德泰拉 物种具有很高的传染性，并会引起类似的临床症状，使它们很难 诊断。虽然副百日咳杆菌感染表现为百日咳杆菌症状较轻- 衍生感染，这两种感染都可能是致命的，特别是对儿童、老年人和 免疫功能受损的病人。目前的诊断方法要么速度慢，要么需要昂贵的费用 乐器。对这两种引起百日咳的低成本护理点(POC)检测 百日咳的快速诊断仍未得到满足，迫切需要病原体 在资源有限的情况下。在我们最近的初步研究中，我们第一次开发了 一种与环介导恒温DNA扩增相结合的低成本微流控技术 (LAMP)用于百日咳杆菌的POC快速检测。在这里，这项建议的目标是发展 一种新型的低成本纸/聚合物混合微流控POC装置，与灯集成在一起， 对两种主要引起百日咳的波尔德氏菌(B. 百日咳和副百日咳杆菌)，可在诸如医生办公室等各种场所使用， 学校和其他资源匮乏的环境。我们的中心假设是低值积分- 成本低廉的纸基微流控技术和特定的DNA检测分析可以提供 在资源匮乏的环境下进行快速、准确和快速的诊断(美国专利#10,875,024)。至 为了实现我们的目标，我们制定了三个具体的目标：(1)开发和优化LAMP 同时检测百日咳杆菌和副百日咳杆菌的方法；(2)建立一种低成本的 纸/聚合物混合微流控POC无仪器同时检测装置 B.百日咳和B.副百日咳；以及(3)验证用于波氏杆菌快速检测的POC设备 使用临床标本的物种。拟议的方法将允许确认 由不同波尔德氏菌属引起的百日咳疑似个案， 填补了目前百日咳诊断方法的空白。此外，我们的生物芯片有很好的 有可能快速检测各种其他呼吸道传染病，如 在资源匮乏的环境下，新冠肺炎。

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.