Direct RT-PCR detection of RNA pathogens in crude samples

描述（由申请人提供）：本申请的目的是介绍一种新的、简化的、低成本的技术，以满足直接在粗样品（如血液）中检测RNA病原体的广泛需求。传统的RT-PCR需要纯化RNA，这增加了成本、时间和交叉污染的风险。我们打算减少或消除纯化步骤，通过生产双功能，热稳定的DNA聚合酶，设计用于直接RT-PCR通过定向诱变我们现有的耐药Omni Klentaq和OmniTaq酶。最有前途的突变酶将作为临床试剂盒的组成部分，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酶中的突变（其赋予对PCR抑制剂的高抗性）与最近发表的突变（其使Taq DNA聚合酶能够逆转录）相结合，进化出更稳健和敏感的双功能酶。作为替代方案，如果在耗尽已公布突变的所有AA取代后，双功能酶的性能不令人满意，我们将使用一种新的和高效的程序对诱变的Taq文库进行功能筛选，以促进工程化独特的双功能酶。实现这一目标的一个关键里程碑是从所选突变体中获得在耐药和RT活性方面的平衡的双重性能。将测试最佳突变酶的抑制抗性、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被优化用于大规模商业质量的酶生产。除了抑制抗性和RT特征之外，所选择的酶应当具有匹配或超过亲本OmniTaq和Omni Klentaq酶的热稳定性和保真度。此外，我们将把我们新的RT-PCR技术应用于临床应用。利用双功能酶或酶的混合物，我们将开发独特、灵敏和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品中的HIV、HCV、GBV-C和登革病毒。我们成功的衡量标准是，我们在粗品临床样本中检测的灵敏度和特异性与现有顶级商业试剂盒对纯化RNA的检测水平相匹配或超过。我们将与我们的合作者在试剂盒的最终验证和营销中协同工作。通过消除PCR之前的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。所提出的方法将提供更高的速度，提高效率，降低成本的RNA检测在重要的临床样本，从而有利于公共卫生。

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.