Development of Novel Hot Start (HS) Reverse Transcriptase Technology

新型热启动（HS）反转录酶技术的开发

• 批准号: 103937
• 金额: $ 44.08万
• 依托单位: PCR BIO SYSTEMS LIMITED
• 依托单位国家: 英国
• 项目类别: Collaborative R&D
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=103937
• 关键词: Development; Novel; Hot; Start; HS

The Polymerase Chain Reaction (PCR) combined with reverse transcription (RT-PCR and qRT-PCR) is a sensitive method to detect RNA and a crucial technique within bioscience and healthcare research, for example in detecting RNA viruses that exist in very low levels in blood and body fluids of infected individuals. However the sensitivity of the technique can be affected by the enzyme used in the process, Reverse Transcriptase (RTase), forming 'primer dimer' and non specific products at lower temperatures during reaction set up. Our research has shown that the current methods used to improve the sensitivity and specificity of the reaction by inhibiting RTase activity at lower temperatures ('hot start') are inferior and do not inhibit RTase activity sufficiently for ultrasensitive RNA detection. We intend to take an innovative approach to RTase inhibition and develop a novel RTase inhibitor and a range of hot start RTase products for cDNA synthesis, RT-PCR and RT-qPCR to dramatically increase the sensitivity and specificity of RNA detection.

聚合酶链式反应（PCR）结合逆转录（RT-PCR和qRT-PCR）是一种检测RNA的灵敏方法，也是生物科学和医疗保健研究中的关键技术，例如检测感染个体血液和体液中极低水平存在的RNA病毒。然而，该技术的灵敏度可能受到该过程中使用的酶（逆转录酶（RTase））的影响，在反应设置期间在较低温度下形成“引物二聚体”和非特异性产物。我们的研究表明，目前用于通过在较低温度下抑制RTase活性（“热启动”）来提高反应灵敏度和特异性的方法是低劣的，并且不能充分抑制RTase活性以进行超灵敏的RNA检测。我们打算采取创新的方法来抑制RTase，并开发一种新的RTase抑制剂和一系列用于cDNA合成、RT-PCR和RT-qPCR的热启动RTase产品，以显著提高RNA检测的灵敏度和特异性。