An Isothermal Method to Amplify RNA from Bloodborne Viruses

扩增血源性病毒 RNA 的等温方法

• 批准号: 10760602
• 负责人: Karl Gorzelnik
• 金额: $ 29.02万
• 依托单位: LYNNTECH, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10760602
• 关键词: Address; Anti-Retroviral Agents; Antibodies; Antigens; Bacterial RNA; Benchmarking; Biological; Biological Assay; Blood; Blood specimen; Buffers; COVID-19 pandemic; DNA Integration; Dedications; Dengue; Detection; Developing Countries; Development; Devices; Diagnostic; Diagnostic Equipment; Ensure; Enzymes; Equipment; General Population; Genome; Guide RNA; HIV-1; Hepatitis A; Hepatitis C; Hepatitis Viruses; Human; Human immunodeficiency virus test; In Situ; Infection; Kinetics; Laboratories; Learning; Measures; Mediating; Methods; Monitor; Nucleic Acid Amplification Tests; Nucleic Acids; Phase; Process; Public Health; RNA; RNA Viruses; RNA amplification; RNA purification; RNA-Directed RNA Polymerase; Reaction; Reproducibility; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Serology test; Signal Transduction; Silicon Dioxide; Small Business Innovation Research Grant; Specificity; System; Testing; Titrations; Validation; Viral; Virion; Virus; Virus Diseases; Virus Replication; Work; ZIKA; amplification detection; antigen detection; assay development; care providers; commercialization; detection limit; diagnostic assay; fighting; improved; isothermal amplification; lateral flow assay; magnetic beads; method development; multiplex detection; novel strategies; nucleic acid detection; particle; pathogenic virus; point of care; point of care testing; point-of-care detection; point-of-care diagnostics; polyacrylate; replicase; risk mitigation; success; usability; vaccine-induced immunity; viral RNA; viral detection

PROJECT SUMMARY Bloodborne RNA viruses, in particular HIV-1 and hepatitis C, are a major public health burden. The diagnostic gold standard for these viruses is the use of RT-qPCR, which is expensive for use in developing countries. Traditional tests for HIV-1 also cannot be used to assess antiretroviral efficacy, as RT-qPCR could pick up integrated DNA and serological tests can detect antigens chromosomally expressed from integrated DNA. We propose the development of a diagnostic assay that will amplify and detect the RNA of bloodborne RNA viruses via a unique RNA amplification process. By developing a method to enrich viral RNAs over eukaryotic or bacterial RNAs we can lower the limit of detection and potentially reduce the level of false positives and negatives observed when using RT-based diagnostics. As this method is specific for viral RNA in general, it can be used for other human ssRNA viral pathogens, such as Zika or Dengue. The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase I project will be the development of a new step in viral RNA detection, usable for detecting the five bloodborne viruses we propose here, but also other ssRNA viruses. By using an isothermal approach to amplify viral RNA our device will eliminate the need for expensive equipment, which will increase access to testing. In the wake of the SARS-CoV-2 pandemic, the general public learned that Point-of-Care (POC) tests for antibodies or antigens do not detect low levels of viral infections. We propose to develop a test that will selectively amplify viral RNAs, which will bring the sensitivity of nucleic acid amplification to POC providers. The proposed work will validate the specificity of the enzyme toward viral RNAs, develop a system to extract and detect these RNAs, and lower the detection limit for POC diagnostics.

项目摘要 血液传播的RNA病毒，特别是HIV-1和丙型肝炎，是一个主要的公共卫生负担。诊断 这些病毒的金标准是使用RT-qPCR，这对于在发展中国家使用来说是昂贵的。 传统的HIV-1检测也不能用于评估抗逆转录病毒的疗效，因为RT-qPCR可以检测出 整合的DNA和血清学测试可以检测从整合的DNA染色体表达的抗原。我们 我建议开发一种诊断方法，可以扩增和检测血液传播的RNA病毒的RNA 通过独特的RNA扩增过程。通过开发一种方法来富集病毒RNA超过真核或 细菌RNA，我们可以降低检测限，并可能降低假阳性和假阴性的水平 在使用基于RT的诊断时观察到。由于该方法通常对病毒RNA具有特异性，因此可以使用 其他人类ssRNA病毒病原体，如寨卡或登革热。更广泛的影响/商业潜力 这个小企业创新研究（SBIR）第一阶段项目将是病毒式发展的新一步， RNA检测，可用于检测我们在这里提出的五种血液传播病毒，也可用于检测其他ssRNA病毒。 通过使用等温方法来扩增病毒RNA，我们的设备将消除对昂贵的 设备，这将增加获得测试。在SARS-CoV-2大流行之后， 公众了解到，针对抗体或抗原的床旁（POC）检测不能检测出低水平的病毒感染。 我们建议开发一种选择性扩增病毒RNA的测试，这将使核酸检测的灵敏度提高。 对POC提供者的酸扩增。这项工作将验证该酶对病毒的特异性 RNA，开发一个系统来提取和检测这些RNA，并降低POC诊断的检测限。