PRESSURE-ASSISTED NUCLEIC ACID EXTRACTION FROM PLASMA

高静水压已被证明在核酸样品制备的许多步骤中非常有用，包括细胞裂解、脱蛋白、核酸酶抑制和从各种生物样品中提取核酸。在第一阶段研究中，将使用BioSeq的创新高压技术检查从血浆中提取RNA的可行性。 HIV 8E5是一种非感染性HIV突变病毒，将用作靶RNA来源。将检测压力、温度和添加必要化学品的曲线，以选择病毒裂解、核酸酶灭活和RNA提取的最佳条件。将对每个步骤进行定性和定量核酸测定，并与常用方法进行比较。 将开发压力提取系统以及提取模块的概念设计和集成的可能性。本研究阶段生成的数据将构成II期研究期间设计用于RNA和DNA分离的快速自动化系统的基础。提出的压力辅助RNA提取将减少苛刻的化学试剂的使用，劳动力和材料成本，以及测定时间。它代表了分子诊断行业在监测传染病方面的重要进展。建议的商业应用：使用高压循环的核酸提取是一种创新工艺。本文提出的从血浆中提取病毒RNA的目的是克服分子诊断中样品制备的一些当前限制。这种压力技术的发展最终将允许快速、自动化的核酸样品制备，而不需要样品的大量稀释或使用刺激性化学品。所提出的技术的成功将不仅有利于病毒诊断，而且有利于更广泛的领域，如传染病诊断，癌症诊断，基因组学和法医分析的研究和临床应用。

High hydrostatic pressure has been demonstrated very useful in many of the steps of nucleic acid sample preparation including cell lysis, deproteination, nuclease inhibition and nucleic acid extraction from various biological samples. During this Phase I study, feasibility of extraction of RNA from plasma will be examined using BioSeq's innovative high-pressure technology. HIV 8E5, a non-infectious HIV mutant virus, will be employed as the target RNA source. Profiles of pressure, temperature, and addition of necessary chemicals will be tested to select the optimum conditions for viral lysis, nuclease inactivation and RNA extraction. Qualitative and quantitative nucleic acid assays will be performed for each step and compared to those from the methods that are commonly used. The pressure extraction system as well as the conceptual design of the extraction module and the possibility of integration will be developed. Data generated in this phase of study will form the basis for the design of a rapid, automated system for RNA and DNA isolation during the Phase II. The proposed pressure-assisted RNA extraction would reduce the usage of harsh chemical reagents, labor and material cost, and assay time. It represents an important advancement in the molecular diagnostics industry for monitoring infectious diseases. PROPOSED COMMERCIAL APPLICATIONS: Nucleic acid extraction using high-pressure cycling is an innovative process. Viral RNA extraction from plasma proposed here aims to overcome some of the current limitations of sample preparation in molecular diagnostics. The development of this pressure technology will ultimately permit rapid, automated nucleic acid sample preparation without the requirement of extensive dilution of the sample or use of harsh chemicals. The success of the proposed technology will benefit not only viral diagnostics, but also broader fields, such as infectious disease diagnostics, cancer diagnosis, genomics and forensic analysis for research and clinical applications.