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

• 批准号: 10430557
• 负责人: WEN-JI DONG
• 金额: $ 18.41万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10430557
• 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 Primers; Detection; Device Designs; Devices; Diagnosis; Diagnostic; Early Diagnosis; Early treatment; Enzymes; Fluorescence; Funding; Generations; Goals; Gold; Guide RNA; Hepatitis B; Hepatitis C; Hepatitis C Antibodies; Hepatitis C Therapy; Hepatitis C virus; Human; Immune; 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; Site; Specificity; Technology; Temperature; Testing; Time; Viral hepatitis; Viremia; Virus Diseases; Visit; antibody test; base; cost; cost effective; density; design; detection limit; detection sensitivity; diagnostic platform; diagnostic technologies; ds-DNA; 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; treatment site; viral RNA

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，肝细胞癌和肝脏的主要原因 移植。随着2013年成功介绍直接作用抗病毒药（DAA），几乎没有 效果，负担得起的价格和> 90％的治疗率用于治疗HCV感染，他们采用了有史以来第一个 到2030年，全球消除病毒肝炎作为公共卫生问题的策略。这个雄心勃勃的目标将需要 重大努力，不仅是为了防止新感染，而且还诊断那些尚未意识到的人 已感染。据估计，在有HCV感染的7100万人中，一半不知道他们 感染，因此未经治疗。向未经处理的人群提供医疗服务的关键，从而 感染的停止传播是在诊断出感染期间的现场立即治疗的 病人定期去医生办公室。这将需要一个可以快速，成本效益的诊断平台 在护理点（POC）使用，并在<30分钟内提供诊断结果。当前的诊断，初始诊断 HCV抗体（AB）测试记录暴露，然后进行更复杂和昂贵的HCV RNA测试 确认病毒血症，无法满足POC的需求。由于血清HCV RNA早在1-2周之前就可以检测到 感染后，基于聚合酶链反应（PCR）RNA分析是HCV的金标准方法 诊断。但是，在POC上实施基于PCR的分析受其缓慢的周转时间的限制 和高成本/功效比率，尤其是在资源贫乏的设置中。该建议旨在填补空白 开发一个新颖的诊断平台，能够通过整合在<20分钟内识别HCV感染 重组酶聚合酶扩增（RPA），CRISPR-CAS12A和带正电荷的金纳米颗粒 （+GNP）基于核酸扩增（NAA）测试的统一的单步测定法（LFA）。 为了实现目标并应对基于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核酸扩增来解决 统一RPA和CRISPR-12A反应中的不相容性问题，2）选择性从充电中运送出来 +CRISPR扩增过程发布的GNP，以解决试剂/酶冲洗问题，3）设计 具有分级目标结合和内在反应“计时器”的新型目标测试线的新数量 与基于LFA的核酸诊断技术相关的问题，以及4）微型基于智能手机 检测，这将使HCV诊断能够兼容POC和移动健康。我们期望成功 该项目的完成将为快速NAA诊断提供一种新颖，强大，具有成本效益的POC技术 HCV感染将对HCV和其他其他治疗的早期诊断和治疗产生广泛的积极影响 传染病。