Direct RT-PCR detection of RNA pathogens in crude samples

描述(申请人提供)：本申请的目的是介绍一种新的、简化的、低成本的技术，以满足直接从粗制样本(如血液)中检测RNA病原体的广泛需求。传统的RT-PCR需要对RNA进行纯化，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耐抑制剂Omni Klentaq和OmniTaq酶进行定向突变，生产双功能、耐热的DNA聚合酶，用于直接RT-PCR，从而减少或消除纯化步骤。最有希望的突变酶将作为临床试剂盒的组成部分，用于检测艾滋病毒、丙型肝炎病毒、GBV-C和登革热病毒。我们还将探索我们的耐抑制酶与逆转录酶的优化混合。首先，我们将通过将我们的Omni Klentaq和OmniTaq酶的突变与最近公布的使Taq DNA聚合酶能够逆转录的突变相结合，进化出更强大和更敏感的双功能酶。作为另一种选择，如果在用尽已公布的突变的所有AA取代后，双功能酶的性能不能令人满意，我们将使用一种新颖而高效的程序来对突变的Taq文库进行功能筛选，以帮助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选定的突变体中获得抑制剂抗性和RT活性的平衡双重表现。最好的突变酶将被测试它们的抗抑制性、逆转录酶活性、敏感性、热稳定性和保真度。最后，将对这些酶的酶纯化方案进行优化，以实现大规模的商业化优质酶生产。除了耐抑制和RT特性外，所选的酶还应具有与亲本OmniTaq和Omni Klentaq酶相匹配或超过的热稳定性和保真度。此外，我们将把我们的新RT-PCR技术应用于临床。利用双功能酶或混合酶，我们将开发独特、灵敏和可靠的单一和多重实时RT-PCR方法，直接检测原始临床样本中的艾滋病毒、丙型肝炎病毒、GBV-C和登革热病毒。我们成功的衡量标准将是我们在粗制临床样本中的检测灵敏度和特异度达到或超过现有顶级商业试剂盒对纯化RNA的检测水平。我们将与我们的合作者共同努力，最终验证和营销这些工具包。通过省去聚合酶链式反应之前的TH RNA提取步骤，所提出的新技术不仅显著降低了成本，而且解决了技术问题。该方法将提高重要临床标本的RNA检测速度、效率和成本，从而有利于公众健康。

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.