Direct RT-PCR detection of RNA pathogens in crude samples

直接 RT-PCR 检测粗样品中的 RNA 病原体

• 批准号: 8715124
• 负责人: Zhian Zhang
• 金额: $ 48.23万
• 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 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8715124
• 关键词: Address; Biological Assay; Biological Markers; Blood; Blood specimen; Catalytic Domain; Centers for Disease Control and Prevention (U.S.); Clinical; Clinical Research; Codon Nucleotides; Collaborations; DNA; DNA-Directed DNA Polymerase; Dengue Virus; Detection; Diagnostic Procedure; Diagnostic tests; Disease; Eligibility Determination; Engineering; Enhancers; Enzymes; Equilibrium; Exhibits; GB virus C; Gene Expression; Gene Targeting; Genes; HIV; HIV-1; Hepatitis; Hepatitis C virus; Human; Infection; Libraries; Marketing; Measures; Methods; Mutagenesis; Mutation; Pathogen detection; Patients; Performance; Performance at work; Phenotype; Plasma; Procedures; Process; Production; Protocols documentation; Provider; Public Health; Publishing; Qualifying; RNA; RNA Viruses; RNA purification; RNA-Directed DNA Polymerase; Reaction; Research; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Risk; Role; Sampling; Sensitivity and Specificity; Serum; Site-Directed Mutagenesis; Specificity; Speed; Structure; System; Taq Polymerase; Technology; Testing; Time; Validation; Viral; Viral Load result; Virus; Virus Diseases; Work; base; clinical application; cost; design; exhaust; improved; inhibitor/antagonist; insight; interest; mutant; new technology; novel; pathogen; public health relevance; screening; success; thermostability; viral RNA

DESCRIPTION (provided by applicant): The objective of this application is to introduce a novel, simplified, low cost technology to address the broad need of detecting RNA pathogens directly in crude samples, such as blood. Traditional RT-PCR requires purifying the RNA which increases the cost, time, and risk of cross-contamination. We intend to reduce or eliminate the purification step by producing bifunctional, thermostable DNA polymerases designed for direct RT-PCR via directed mutagenesis of our existing inhibitor-resistant Omni Klentaq and OmniTaq enzymes. The most promising mutant enzymes will serve as components in clinical kits for detection of HIV, HCV, GBV-C, and dengue virus. We will also explore optimized blends of our inhibition-resistant enzymes with reverse transcriptases. First, we will evolve more robust and sensitive bifunctional enzymes by combining the mutations in our Omni Klentaq and OmniTaq enzymes that confer high resistance to PCR inhibitors with recently published mutations rendering Taq DNA polymerase capable of reverse transcription. As an alternative, if after exhausting all AA substitutions of the published mutations the performance of the bifunctional enzymes is not satisfactory, we will use a novel and highly efficient procedure for functional screening of mutagenized Taq libraries to facilitate engineer the unique bifunctional enzymes. A critical milestone in achieving this aim is to obtain balanced dual performance from the selected mutants in inhibitor-resistance and RT activity. The best mutant enzymes will be tested for their inhibition-resistance, reverse transcriptase activity, sensitivity, thermostability, and fidelity. Finally, the enzyme purification protocol for these enzymes will be optimized for large-scale commercial quality enzyme production. Besides inhibition-resistance and RT features, the selected enzymes should have thermostability and fidelity matching or exceeding that of the parental OmniTaq and Omni Klentaq enzymes. Further, we will apply our novel RT-PCR technology to clinical applications. With bifunctional enzymes or blends of enzymes, we will develop unique, sensitive, and reliable single and multiplex real-time RT-PCR assays for direct detection of HIV, HCV, GBV-C, and dengue virus in crude clinical samples. Our measure of success will be that the sensitivity and specificity of our assays in crude clinical samples match or exceed the detection level of existing top commercial kits on purified RNA. We will work in concert with our collaborators in the final validation and marketing of the kits. By eliminating th RNA extraction steps prior to PCR, the proposed novel technology not only introduces a significant reduction in cost, but also solves technical problems. The proposed method would provide higher speed, improved efficiency, and lower cost of RNA detection in important clinical samples, thereby benefitting public health.

描述（由申请人提供）：本申请的目的是引入一种新颖、简化、低成本的技术，以满足直接检测粗样品（例如血液）中RNA病原体的广泛需求。传统 RT-PCR 需要纯化 RNA，这增加了成本、时间和交叉污染的风险。我们打算通过对我们现有的抗抑制剂 Omni Klentaq 和 OmniTaq 酶进行定向诱变，生产专为直接 RT-PCR 设计的双功能热稳定 DNA 聚合酶，从而减少或消除纯化步骤。最有前途的突变酶将作为临床试剂盒的成分，用于检测 HIV、HCV、GBV-C 和登革热病毒。我们还将探索抗抑制酶与逆转录酶的优化混合物。首先，我们将通过将 Omni Klentaq 和 OmniTaq 酶中赋予 PCR 抑制剂高抗性的突变与最近发表的使 Taq DNA 聚合酶能够逆转录的突变相结合，进化出更强大、更灵敏的双功能酶。作为替代方案，如果在用尽已发表突变的所有 AA 取代后，双功能酶的性能仍不令人满意，我们将使用一种新颖且高效的程序对诱变 Taq 文库进行功能筛选，以方便设计独特的双功能酶。实现这一目标的一个关键里程碑是从所选突变体中获得抑制剂抗性和 RT 活性的平衡双重性能。将测试最好的突变酶的抑制抗性、逆转录酶活性、敏感性、热稳定性和保真度。最后，这些酶的酶纯化方案将针对大规模商业质量酶生产进行优化。除了抑制抗性和 RT 特性外，所选酶还应具有与亲本 OmniTaq 和 Omni Klentaq 酶相匹配或超过的热稳定性和保真度。此外，我们将把我们新颖的RT-PCR技术应用于临床应用。借助双功能酶或酶混合物，我们将开发独特、灵敏且可靠的单一和多重实时 RT-PCR 检测方法，用于直接检测粗临床样本中的 HIV、HCV、GBV-C 和登革热病毒。我们衡量成功的标准是，我们在粗临床样品中进行的检测的灵敏度和特异性符合或超过现有顶级商业试剂盒对纯化 RNA 的检测水平。我们将与合作者共同完成套件的最终验证和营销。通过消除 PCR 之前的 RNA 提取步骤，所提出的新技术不仅显着降低了成本，而且解决了技术问题。所提出的方法将为重要临床样本中的RNA检测提供更高的速度、更高的效率和更低的成本，从而有利于公众健康。