Smartphone-linked system for diagnosis and epidemiological reporting of pathogens at the point of care

摘要 传染病仍然是世界上造成死亡和残疾的最主要原因， 快速通过昆虫媒介或与体液接触，迫切需要简单、敏感和 易于翻译的护理点测试，特别是对于难以区分的感染 仅临床症状。该项目开发了一种新的床旁平台，用于定量诊断病毒 感染性疾病（寨卡病毒、登革热（1型和3型）和基孔肯雅病毒）， 微流控平台，其在微流控盒中执行样品预处理，随后进行实时 逆转录环介导的等温扩增（RT-LAMP）在同一盒中与预干燥的 特异于病原体靶标的引物。我们的手持式，廉价（约500美元），即时护理平台， 将其测量结果发送到智能手机，以处理扩增反应的荧光图像序列。 动态扩增过程的图像分析用于估计来自扩增的病原体计数 使用称为“空间”LAMP（S-LAMP）的新方法来检测起始点。初步数据显示， 该方法提供的选择性和检测限与传统聚合酶链反应相当 使用常规实验室仪器进行的PCR反应（PCR）或LAMP反应， 从复杂介质中检测出高特异性的靶标。S-LAMP方法证明了 每个反应检测1-5个病原体拷贝。该系统将采用严格的分层方法进行评估， 含有靶核酸序列的质粒，用于检测限的初始表征，然后 核酸病原体提取物，最终使用化学裂解检测全血中的病原体 以释放靶核酸。与巴西传染病研究所的合作者合作， 该系统将用于患者样本库和测量 与黄金标准分析相比。重要的是，由于检测、图像处理和定量是 通过智能手机微处理器执行，检测很容易与基于云的数据库集成 报告系统，便于与远程医生进行通信，并跟踪 卫生部门的流行病学数据。由此产生的平台将广泛适用于各种各样的 对人类健康、动物健康、食品安全感兴趣的病原体的多重组， 环境监测

Abstract Infectious disease remains the world’s top contributor to death and disability, and, due to their ability to spread rapidly through insect vectors or contact with bodily fluids, there is an urgent need for simple, sensitive and easily translatable point-of-care tests, particularly for infections that are difficult to differentiate based upon clinical symptoms alone. This project develops a novel point-of-care platform to quantitatively diagnose viral infectious diseases (Zika, Dengue (types 1 and 3), and Chikungunya) from whole blood samples using a microfluidic platform that performs sample pre-processing in a microfluidic cartridge followed by real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) in the same cartridge with pre-dried primers specific to pathogen targets. Our handheld, inexpensive (~$500), point-of-care platform communicates its measurements to a smartphone to process a sequence of fluorescence images of the amplification reaction. Image analysis of the dynamic amplification process is used to estimate the pathogen count from amplification initiation points using a novel approach called “spatial” LAMP (S-LAMP). Our preliminary data shows that the approach provides selectivity and detection limits that are equivalent to conventional Polymerase Chain Reaction (PCR) or LAMP reactions performed with conventional laboratory instruments, and the ability to detect the targets with high specificity from complex media. The S-LAMP approach demonstrates potential to detect 1-5 pathogen copies per reaction. The system will be evaluated using a rigorous tiered approach using plasmids containing the target nucleic acid sequence for initial characterization of detection limits, followed by nucleic acid pathogen extracts, culminating of detection of the pathogens in whole blood using chemical lysis to release target nucleic acids. Partnering with collaborators at the Institute for Infectious Diseases in Brazil, and Carle Clinic in Urbana, the system will be utilized on patient sample repositories and measurements compared to gold standard analysis. Importantly, as detection, image processing, and quantitation are performed with the smartphone microprocessor, detection is easily integrated with a cloud-based database reporting system that facilitates communication with remotely-located physicians and tracking of epidemiological data by health services. The resulting platform will be broadly applicable to a wide variety of multiplexed panels of pathogens that are of interest for human health, animal health, food safety, and environmental monitoring.