Direct RT-PCR detection of RNA pathogens and mRNA expression in crude samples

直接 RT-PCR 检测粗样品中 RNA 病原体和 mRNA 表达

• 批准号: 7911473
• 负责人: Zhian Zhang
• 金额: $ 10.59万
• 依托单位: DNA POLYMERASE TECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7911473
• 关键词: Amino Acid Substitution; Biological Assay; Blood; Blood specimen; Buffers; Cells; Clinical; Complementary DNA; Cytolysis; DNA; DNA-Directed DNA Polymerase; Detection; Development; Direct Costs; Enhancers; Enzymes; Evaluation; Gene Expression; Goals; Government; HIV; Hepatitis; Hepatitis C virus; Hereditary Disease; Human; Influenza; Measures; Methods; Nucleic Acids; Pathogen detection; Patients; Performance; Persons; Plasma Cells; Procedures; Process; Protocols documentation; RNA; RNA Viruses; RNA purification; RNA-Directed DNA Polymerase; Reaction; Research; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Sampling; Serum; Site-Directed Mutagenesis; Specimen; System; Technology; Testing; Time; Tissues; Viral; Virus; Whole Blood; Work; base; clinical application; communicable disease diagnosis; cost; design; improved; inhibitor/antagonist; mRNA Expression; mutant; new technology; novel; pathogen; public health relevance

DESCRIPTION (provided by applicant): We propose to develop a highly simplified and improved method of detecting RNA for use in clinical tests and for scientific research by enabling the RT-PCR amplification of nucleic acids directly in whole blood, serum, plasma, and cell lysates. We propose a dual approach. We will work with two of our blood inhibition Taq mutants combined with a viral reverse transcriptase enzyme in the presence of a specially developed enhancer. In addition, we will make amino acid substitutions to our blood inhibition mutants to render them competent in reverse transcription. The sensitivity of the new technology will drive the evaluations. The method also includes development and optimization of buffers that are compatible with both the RT and DNA polymerase activities, as well as reaction additives that relieve the inhibition and enhance the RT-PCR performance in crude specimens. The tests will begin with mimic samples composed of RNA mixed with blood, serum, and plasma. Once optimized, the method will be applied to clinical RNA pathogen detection and mRNA expression assays in crude samples. The clinical RNA virus pathogens will include HCV and GBV. Comparisons will be made to the standard RNA detection protocol, which requires the RNA to be purified from the sample prior to detection. The focus of the novel method will be to increase the sensitivity of detection to match or exceed the sensitivity of the standard method. Until now, the diagnosis of infectious diseases and genetic disorders has required costly and time-consuming procedures. The standard protocol requires RNA purification prior to RT-PCR which may reduce the quantity of RNA before cDNA is produced. The proposed novel technology not only introduces a significant reduction in cost, but also solves technical problems irrespective of cost. The proposed method would provide improved accuracy, efficiency, and lower cost of RNA detection directly in whole blood or blood fractions samples and cell and tissue lysates. The benefit to the public is by improved and more reliable detection of RNA pathogens in clinical tests and advanced means of measuring mRNA expression in crude samples at a reduced cost. PUBLIC HEALTH RELEVANCE: Many viruses that are harmful to humans, such as hepatitis, HIV, and influenza, are based in RNA, not DNA. To determine if a patient has a harmful RNA virus, a blood sample is often drawn then sent to a lab to use a process called RT-PCR to find the virus. Until now, for RT- PCR to work, the RNA must first be extracted from the blood which can present problems and is expensive, but our proposed method can find RNA directly in blood.

描述（由申请人提供）：我们建议开发一种高度简化和改进的 RNA 检测方法，通过直接在全血、血清、血浆和细胞裂解物中进行核酸的 RT-PCR 扩增，用于临床测试和科学研究。我们提出双重方法。我们将在专门开发的增强剂存在下将两种血液抑制 Taq 突变体与病毒逆转录酶结合使用。此外，我们将对我们的血液抑制突变体进行氨基酸取代，以使它们能够进行逆转录。新技术的敏感性将推动评估。该方法还包括开发和优化与 RT 和 DNA 聚合酶活性兼容的缓冲液，以及可缓解抑制并增强粗样品中 RT-PCR 性能的反应添加剂。测试将从由 RNA 与血液、血清和血浆混合组成的模拟样本开始。经过优化后，该方法将应用于临床 RNA 病原体检测和粗样品中 mRNA 表达测定。临床RNA病毒病原体将包括HCV和GBV。将与标准 RNA 检测方案进行比较，后者要求在检测前从样品中纯化 RNA。新方法的重点是提高检测的灵敏度，以达到或超过标准方法的灵敏度。到目前为止，传染病和遗传性疾病的诊断需要昂贵且耗时的程序。标准方案要求在 RT-PCR 之前纯化 RNA，这可能会在产生 cDNA 之前减少 RNA 的数量。所提出的新技术不仅显着降低了成本，而且解决了与成本无关的技术问题。所提出的方法将提高直接在全血或血液成分样品以及细胞和组织裂解物中进行 RNA 检测的准确性、效率并降低成本。对公众的好处是在临床试验中改进和更可靠地检测 RNA 病原体，以及以更低的成本测量粗样品中 mRNA 表达的先进方法。 公共卫生相关性：许多对人类有害的病毒，如肝炎、艾滋病毒和流感，都是基于 RNA，而不是 DNA。为了确定患者是否感染了有害的 RNA 病毒，通常会抽取血液样本，然后送往实验室，使用 RT-PCR 的过程来查找病毒。到目前为止，为了使 RT-PCR 发挥作用，必须首先从血液中提取 RNA，这可能会带来问题并且价格昂贵，但我们提出的方法可以直接在血液中找到 RNA。