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

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，用于临床测试和科学研究，方法是能够直接从全血、血清、血浆和细胞裂解物中扩增核酸。我们提出了一种双重方法。我们将在一种特殊开发的增强剂的存在下，将我们的两个血液抑制Taq突变体与病毒逆转录酶相结合。此外，我们将对我们的血液抑制突变体进行氨基酸替换，使其能够进行逆转录。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，以及在粗制样品中解除抑制和提高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。一旦优化，该方法将应用于临床RNA病原体检测和粗样品中的mRNA表达分析。临床RNA病毒病原体将包括丙型肝炎病毒和GBV。将与标准的RNA检测方案进行比较，后者要求在检测之前从样本中提纯RNA。新方法的重点将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和耗时的程序。标准的方法要求在RT-PCR之前对RNA进行纯化，这可能会在产生cDNA之前减少RNA的数量。建议的新技术不仅大大降低了成本，而且解决了不计成本的技术问题。所提出的方法将提供更高的准确性、效率和更低的成本，直接在全血或血液部分样本以及细胞和组织裂解物中检测RNA。对公众的好处是在临床测试中改进和更可靠地检测RNA病原体，以及以更低的成本测量原始样本中的mRNA表达的先进手段。 与公共卫生相关：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，通常会抽取血样，然后送往实验室，使用一种名为RT-PCR的过程来检测病毒。到目前为止，要使RT-PCR发挥作用，必须首先从血液中提取RNA，这可能会出现问题，而且成本很高，但我们提出的方法可以直接在血液中检测到RNA。

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.