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

使用创新的集成注射器直接从全血中进行多重 rRT-PCR 检测寨卡病毒、登革热和基孔肯雅热

• 批准号: 9807185
• 负责人: Brian N Kim
• 金额: $ 21.62万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9807185
• 关键词: 3-Dimensional; Automation; Biochemical; Biological Assay; Bite; Blood; Blood specimen; Cessation of life; Chikungunya virus; Clinics and Hospitals; Collection; Communicable Diseases; Congenital Abnormality; Culicidae; Cytolysis; Dengue; Dengue Infection; Dengue Virus; Detection; Development Plans; Devices; Diagnosis; Diagnostic; Disease; Doctor of Medicine; Early Diagnosis; Electricity; Electronics; Fluorescence; Freeze Drying; Gold; Hour; Infection; Laboratories; Laboratory Diagnosis; Logistics; Measures; Medical; Methods; Mosquito-borne infectious disease; Motion; Neurologic; Nucleic Acids; Optics; Outcome; Pathogenicity; Patients; Performance; Persons; Plug-in; Point-of-Care Systems; Polymerase Chain Reaction; Population; Preparation; Printing; Procedures; Process; Production; RNA; Reagent; Research Personnel; Sampling; Severities; Sexual Transmission; Silicon Dioxide; Syringes; System; Testing; Time; Training; Translating; Transportation; Tube; United States; Vacuum; Weight; Whole Blood; Work; ZIKV infection; Zika Virus; absorption; amplification detection; base; chikungunya; communicable disease diagnosis; cost; design; experience; global health; improved; innovation; iterative design; multiplex detection; nucleic acid detection; nucleic acid purification; operation; pathogen; portability; prevent; process optimization; sample collection; tool; viral RNA; viral detection

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.

项目摘要/摘要 诊断蚊子传播疾病，例如寨卡病毒（Zikv），登革热病毒（DENV）和Chikungunya病毒 （chikv），在感染的早期阶段，对于限制人口之间的传播至关重要（通过 被感染的蚊子和性传播），并防止患者严重疾病。许多早期 - 近年来已经开发了舞台诊断设备，以检测具有提高敏感性和的病原体 更简单/更便宜的设备，但它们无法改善医学后勤系统以早期诊断感染力 疾病，由于劳动密集型样本准备过程。简单，低成本，健壮的样本收集和 当前需要不需要电力或熟练劳动的样本处理工具，以便早期诊断 传染病。 我们建议设计和制造一个新的血液采样注射器，该注射器完全掺入核酸 纯化功能。这个创新的想法使用了柱塞在注射器中产生的真空 执行血液裂解，二氧化硅吸收，洗涤和稳定任务。如果成功，请简单地抽血 患者将导致准备核酸检测的完全准备好的样品。另外，一个小的 手持和可靠的实时聚合酶链反应（PCR）设备，能够多重检测 Zikv，Denv和Chikv将开发直接与集成注射器合作。实时PCR 设备将被设计为在没有外部电源的情况下工作几个小时，并且可以通过插入易于操作 集成的注射器和墨盒。 ZIKV，DENV和CHIKV的多路复用检测将由 自动激发/发射滤波器炮塔，该设备可以分析核酸扩增到 确定样品中病原核酸的量，以揭示感染的阶段。便携式， 手持实时PCR将使用低成本材料制造，以低成本的大规模生产和 启用大量设备。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，由集成注射器组成 时间PCR设备和墨盒。该平台是通过简单地使用患者从患者那里吸血来操作的 集成注射器并将注射器插入实时PCR设备，从而导致转移 从注射器到墨盒的纯化病毒RNA。墨盒将冻干的实时PCR试剂固定到 RNA放入墨盒中后，立即使核酸扩增能够放大。两步诊断平台 它具有全面复制标准感染性疾病诊断过程的能力独特 目前在诊所和医院中使用，并且可以通过注射器中的样品制备自动化来实现。 拟议的项目可能会对全球健康产生变革性影响，以处理感染性 通过实现早期诊断来诊断疾病。