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.

摘要