Multiplexed rRT-PCR detection of Zika, dengue, and chikungunya directly from whole blood using an innovative integrated syringe

项目总结/摘要 诊断蚊媒疾病，如寨卡病毒（ZIKV）、登革热病毒（DENV）和基孔肯雅病毒 （CHIKV），在感染的早期阶段，对于限制人群中的传播至关重要（通过叮咬， 受感染的蚊子和性传播），以及防止病人患上严重疾病。许多早期- 近年来已经开发了分级诊断装置以检测具有改进的灵敏度的病原体， 更简单/更便宜的设备，但它们未能改善用于感染性疾病早期诊断的医疗后勤系统。 疾病，由于劳动密集型样品制备过程。简单、低成本、可靠的样品采集， 目前需要不需要动力或熟练劳动力的样品处理工具，以能够早期诊断 传染病 我们建议设计和制造一种新的血液采样注射器， 净化功能这个创新的想法利用注射器中的柱塞产生的真空， 执行血液溶解、二氧化硅吸收、洗涤和稳定任务。如果成功的话，一个简单的抽血 患者将得到准备好用于核酸检测的完全制备的样品。另外，一个小的， 手持式、稳健的实时聚合酶链反应（PCR）装置，能够多重检测 将开发ZIKV、DENV和CHIKV，以直接与集成注射器配合使用。实时PCR 设备将被设计为在没有外部电源的情况下工作数小时，并且通过插入 所述一体式注射器和药筒。ZIKV、DENV和CHIKV的多重检测将通过以下方式实现： 自动激发/发射滤光器转台，并且该装置可以分析核酸扩增， 确定样本中病原核酸的数量以揭示感染阶段。便携式， 手持实时PCR将用低成本材料制造，以允许低成本大规模生产， 能够实现设备的大分布。每个实时PCR设备的估计材料成本约为300美元。 该项目将产生一个全面的两步诊断平台，包括一个集成的注射器，真实的- 时间PCR装置和试剂盒。该平台的操作是通过简单地从患者身上抽血， 将注射器插入实时PCR装置中，这导致 将纯化的病毒RNA从注射器注入药筒中。检测盒可容纳冻干的实时PCR试剂， 一旦RNA洗脱到检测盒中，就能够进行核酸扩增。两步诊断平台 具有全面复制标准传染病诊断过程的能力， 目前在诊所和医院中使用，并且通过注射器中的样品制备的自动化来实现。 拟议的项目可能会对全球卫生在处理传染病方面产生变革性影响。 通过早期诊断进行疾病诊断。

PROJECT SUMMARY/ABSTRACT Diagnosing mosquito-borne diseases, such as Zika virus (ZIKV), dengue virus (DENV), and chikungunya virus (CHIKV), in the early stage of infection is crucial in limiting the spread among the population (through bites of infected mosquitos and sexual transmission) as well as preventing serious illness to the patient. Many early- stage diagnostic devices have been developed in recent years to detect pathogens with improved sensitivity and simpler/cheaper devices, but they fail to improve the medical logistical system for early diagnosis of infectious disease, due to the labor-intensive sample preparation process. Simple, low-cost, robust sample collection and sample processing tools requiring no power or skilled labor are currently in demand to enable early diagnosis of infectious disease. We propose to design and fabricate a new blood-sampling syringe that fully incorporates a nucleic acid purification function. This innovative idea uses the vacuum created by the plunger in the syringe to continuously perform blood lysis, silica absorption, washing, and stabilization tasks. If successful, a simple draw of blood from patients will result in a completely prepared sample that is ready for nucleic acid detection. Also, a small, handheld, and robust real-time polymerase chain reaction (PCR) device, capable of multiplexed detection of ZIKV, DENV, and CHIKV, will be developed to work directly with the integrated syringe. The real-time PCR device will be designed to work without external power for several hours and is simple to operate by inserting the integrated syringe and a cartridge. The multiplexed detection of ZIKV, DENV, and CHIKV will be enabled by the automated excitation/emission filter turret, and the device can analyze nucleic acid amplifications to determine the quantity of pathogenic nucleic acid in the sample to reveal the stage of infection. The portable, handheld real-time PCR will be fabricated with low-cost materials to allow mass production at low cost and enable a large distribution of the device. The estimated material cost for each real-time PCR device is ~300 USD. This project will produce a comprehensive two-step diagnosis platform, consisting of an integrated syringe, real- time PCR device, and cartridges. The platform is operated by simply drawing blood from a patient using the integrated syringe and inserting the syringe into the real-time PCR device, which results in a transferring of purified viral RNAs from the syringe into the cartridge. The cartridge holds lyophilized real-time PCR reagents to enable nucleic acid amplification as soon as the RNA is eluted into the cartridge. The two-step diagnosis platform is unique in its capability of comprehensively replicating the standard infectious disease diagnosis process, currently used in clinics and hospitals, and is enabled by the automation of sample preparation in the syringe. The proposed project could potentially make a transformative impact to global health in handling infectious disease diagnosis by enabling early-stage diagnostics.