Paper-Based Nucleic Acid Amplification Test for Rapid Diagnosis of Hepatitis C Viral Infection

纸基核酸扩增测试快速诊断丙型肝炎病毒感染

基本信息

批准号：
10558611
负责人：
WEN-JI DONG
金额：
$ 22.24万
依托单位：
WASHINGTON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10558611
关键词：
Acute Hepatitis Address Adopted Antiviral Agents Awareness Binding Biological Assay Blood specimen Caring Cellular Phone Charge Chronic Hepatitis C Cirrhosis Clinical Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Color Communicable Diseases Complex Continuous Capillary Coupling DNA DNA Primers Detection Device Designs Devices Diagnosis Diagnostic Early Diagnosis Early treatment Enzymes Fluorescence Funding Generations Goals Guide RNA Hepatitis B Hepatitis C Hepatitis C Antibodies Hepatitis C Therapy Hepatitis C virus Human Immune Incubated Infection Measures Medical Membrane Methods Monitor Nucleic Acid Amplification Tests Nucleic Acids Paper Patients Persons Phase Point of Care Technology Polymerase Polymerase Chain Reaction Population Price Primary carcinoma of the liver cells Process Public Health RNA RNA analysis RNA primers Reaction Reaction Time Reagent Reporting Resource-limited setting Sampling Scheme Sensitivity and Specificity Signal Transduction Single-Stranded DNA Site Specificity Technology Temperature Testing Time Viral hepatitis Viremia Virus Diseases Visit With laterality antibody test cost cost effective density design detection limit detection sensitivity diagnostic platform diagnostic technologies ds-DNA efficacy evaluation improved innovation lateral flow assay liver transplantation mHealth miniaturize nanoGold novel novel diagnostics point of care point-of-care diagnostics prevent prototype rapid diagnosis recombinase side effect success technology development technology platform technology validation treatment site viral RNA

项目摘要

Abstract Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis, hepatocellular carcinoma, and liver transplantation. With the successful introduction in 2013 of a direct-acting antiviral (DAA) drug with few side effects, an affordable price and a >90% cure rate for the treatment of HCV infection, WHO adopted the first-ever global strategy for eliminating viral hepatitis as a public health problem by 2030. This ambitious goal will require major efforts, not only to prevent new infections but also to diagnose those who are not yet aware of being infected. It is estimated that of the 71 million people who are living with HCV infection, half are unaware of their infection and are thus untreated. A key to providing medical care to this untreated population, and thus to stopping spread of the infection, is to initiate immediate treatment on site once the infection is diagnosed during a patient's regular visit to a doctor's office. This will require a rapid, cost-effective diagnostic platform that can be used at the point of care (POC) and provide diagnostic results in <30 minutes. The current diagnostics, an initial HCV antibody (Ab) test to document exposure followed by the more complex and expensive HCV RNA test to confirm viremia, cannot meet the POC need. Since serum HCV RNA can be detected as early as 1-2 weeks after infection, polymerase chain reaction (PCR) based RNA analysis is the gold standard method for HCV diagnosis. However, implementation of PCR-based analysis at the POC is limited by its slow turnaround time and high cost/efficacy ratio, especially in resource-poor settings. This proposal is aimed at filling the gap by developing a novel diagnostic platform capable of identifying HCV infection in <20 minutes by integrating recombinase polymerase amplification (RPA), CRISPR-Cas12a and a positively charged gold nanoparticle (+GNP) based lateral flow assay (LFA) into a unified single-step assay for nucleic acid amplification (NAA) test. To achieve the objective and address the technology challenges facing an LFA-based POC NAA test, this project will implement several principal innovations: 1) use of RNA primers for RPA nucleic acid amplification to address the incompatibility issue in unifying RPA and CRISPR-12a reactions, 2) selective transporting out of the charged +GNPs released by the CRISPR amplification process to address the reagent/enzyme washout issue, 3) design of novel target test lines with graded target-binding and an intrinsic reaction “timer” to address the quantification issue associated with LFA-based nucleic acid diagnostic technology, and 4) miniaturized smartphone-based detection, which will make HCV diagnosis more POC and mobile health compatible. We expect that successful completion of this project will lead to a novel, robust, cost-effective POC technology for the rapid NAA diagnosis of HCV infection, which will have broad positive impacts on the early diagnosis and treatment of HCV and other infectious diseases.

抽象的慢性丙型肝炎病毒（HCV）感染是肝硬化、肝细胞癌和肝癌的主要原因 2013 年成功推出副作用少的直接作用抗病毒 (DAA) 药物。世界卫生组织采用了首个治疗丙型肝炎病毒感染的方法，效果显着，价格实惠，治愈率>90% 到 2030 年消除病毒性肝炎这一公共卫生问题的全球战略。这一雄心勃勃的目标需要重大，不仅是为了预防新的感染，也是为了诊断那些尚未意识到感染的人据估计，在 7100 万 HCV 感染者中，有一半人不知道自己感染了 HCV。感染并因此得不到治疗，这是向这些未经治疗的人群提供医疗护理的关键，从而也是阻止感染传播，是指一旦在感染期间确诊感染，立即开始现场治疗患者定期去医生办公室就诊，这需要一个快速、经济高效的诊断平台。在护理点 (POC) 使用并在 30 分钟内提供诊断结果当前诊断，初步。 HCV 抗体 (Ab) 测试可记录暴露情况，随后进行更复杂和昂贵的 HCV RNA 测试，以记录暴露情况确认病毒血症，无法满足 POC 需求，因为血清 HCV RNA 最早可在 1-2 周内检测到。感染后，基于聚合酶链式反应 (PCR) 的 RNA 分析是 HCV 的金标准方法然而，基于 PCR 的分析在 POC 的实施因其周转时间缓慢而受到限制。成本/效益比高，特别是在资源匮乏的地区。该提案旨在通过以下方式填补这一空白。开发一种新型诊断平台，能够通过集成在 20 分钟内识别 HCV 感染重组酶聚合酶扩增 (RPA)、CRISPR-Cas12a 和带正电的金纳米颗粒 (+GNP) 基于侧向层析测定 (LFA) 的核酸扩增 (NAA) 测试的统一单步测定。为了实现这一目标并解决基于 LFA 的 POC NAA 测试面临的技术挑战，该项目将实施几项主要创新：1）使用RNA引物进行RPA核酸扩增，以解决统一 RPA 和 CRISPR-12a 反应的不兼容问题，2) 选择性转运出带电 + CRISPR 扩增过程释放的 GNP，用于解决试剂/酶冲洗问题，3) 设计具有分级目标结合和内在反应“计时器”的新型目标测试线，以解决定量问题与基于 LFA 的核酸诊断技术相关的问题，以及 4) 基于小型化智能手机的检测，这将使 HCV 诊断更加兼容 POC 和移动健康，我们预计会成功。该项目的完成将带来一种新颖、强大、具有成本效益的 POC 技术，用于快速 NAA 诊断 HCV感染的发生，将对HCV及其他疾病的早期诊断和治疗产生广泛的积极影响传染病。