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

项目摘要/摘要 诊断蚊媒疾病，如寨卡病毒、登革热病毒和基孔肯雅病毒 (CHIKV)，在感染的早期阶段对于限制在人群中的传播至关重要(通过叮咬 受感染的蚊子和性传播)，以及防止患者患上严重疾病。很多人很早就- 近年来已经开发出分期诊断设备，以提高检测病原体的灵敏度和 更简单/更便宜的设备，但它们未能改善早期诊断传染病的医疗后勤系统 疾病，由于样品制备过程中的劳动密集型。简单、低成本、可靠的样本收集和 目前需要不需要动力或熟练劳动力的样品处理工具，以实现早期诊断 传染病。 我们建议设计和制造一种新的血液采样注射器，它完全包含一种核酸 净化功能。这一创新的想法利用注射器中柱塞产生的真空来持续 执行血液溶解、二氧化硅吸收、洗涤和稳定任务。如果成功，只需简单地从 患者将得到一个完全准备好的样本，准备好进行核酸检测。还有，一个小的， 手持、强大的实时聚合酶链式反应(PCR)设备，能够多路检测 ZIKV、DENV和CHIKV将被开发为直接与集成注射器一起工作。实时定量聚合酶链式反应 该设备将被设计为在没有外部电源的情况下工作几个小时，并通过插入 一体式注射器和一个弹壳。将通过以下方式启用ZIKV、DENV和CHIKV的多路传输检测 自动激励/发射过滤器转塔，该设备可以分析核酸扩增以 测定样本中致病核酸的数量，以揭示感染的阶段。便携的， 手持式实时聚合酶链式反应将用低成本材料制造，以便以低成本和 启用设备的大范围分发。每个实时聚合酶链式反应装置的估计材料成本约为300美元。 该项目将生产一个全面的两步诊断平台，包括一个集成的注射器，真正的 时间聚合酶链式反应装置和墨盒。该平台只需从患者身上抽血即可 集成注射器并将注射器插入实时聚合酶链式反应装置，从而导致转移 从注射器中提纯的病毒RNA进入试剂盒。该盒保存冷冻干燥的实时PCR试剂以 一旦RNA被洗脱到试剂盒中，就启动核酸放大。两步诊断平台 其独特之处在于其全面复制标准传染病诊断程序的能力， 目前在诊所和医院使用，并通过注射器中样本准备的自动化实现。 拟议的项目可能会在应对传染病方面对全球卫生产生变革性的影响。 通过启用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.