Rapid Microbial Identification by MALDI-TOF Mass Spectrometry of Ribosomal RNA

描述(申请人提供)：世界卫生组织估计，每小时有1600多人死于传染病，其中一半是5岁以下的儿童。传染病占全球总死亡率的26%，是美国第三大死因。除了用于临床反应的微生物鉴定外，诊断还需要用于广泛的应用，包括食品和水安全、生物反应器分析、制药业的无菌保证、环境微生物学和国土安全。本研究的重点是开发和表征一种微生物鉴定平台，该平台使用非常快速的修饰核糖体RNA片段的MALDI-TOF质谱仪。通过产生针对质谱学特征进行优化的碱基特定RNA片段，我们已经证明，可以在大约1.5小时内识别出单一菌落和克隆等“生物纯”样本。该平台是“开放的”，因为它可以识别任何以前已经测序的细菌有机体，而不需要事先设计序列特定的核酸探针。此外，该分析比核酸测序更快，并且能够识别生物混合物中的生物并确定它们的相对丰度。最后，病原体可以在无趣的共生生物体或宿主核酸的背景下被识别。我们计划通过改进基于相对生物丰度的核酸分离技术以及生物混合物分析的启发式方法，来构建我们有希望的通用细菌鉴定原型系统。这项研究的预期结果将是一个快速、第一反应系统，能够对各种类型的样本进行高通量微生物群落分析。包括核酸提取、数据采集和分析，鉴定(S)应该在不到1.5小时内可行。 对传染病的快速反应要求所采用的分析方法本身就是快速的。与大多数分析技术相比，质谱学通常更快，同时提供复杂混合物的分离和基本测量。除了用于临床反应的微生物鉴定外，诊断还需要用于广泛的应用，包括食品和水安全、生物反应器分析和无菌保证以及环境微生物学。该项目的重点是利用预测软件、新的分子技术和核糖体RNA片段的质谱分析来开发一个完整的微生物鉴定系统。

DESCRIPTION (provided by applicant): The World Health Organization estimates that over 1,600 people die each hour from an infectious disease, half of whom are children under 5 years of age. Infectious diseases account for 26 percent of total global mortality and are the third leading cause of death in the United States. In addition to microbial identification for clinical response, diagnostics are also needed for a wide range of applications including food and water safety, bioreactor analysis, sterility assurance in the pharmaceutical industry, environmental microbiology, and homeland security. The focus of this research is to develop and characterize a microbial identification platform using very rapid MALDI-TOF mass spectrometry of modified ribosomal RNA fragments. By generating base-specific RNA fragments optimized for mass spectrometric characterization, we have shown that "organism-pure" samples such as single colonies and clones can be identified in ca. 1.5 hours. The platform is "open" in that it can identify any bacterial organism that has been previously sequenced without the a priori design of sequence specific nucleic acid probes. Furthermore, the assay is more rapid than nucleic acid sequencing and amenable to identification of organisms in biological mixtures and determination of their relative abundances. Finally, pathogens can be identified against an uninteresting background of commensal organisms or host nucleic acids. We plan to build on our promising prototype system for general bacterial identification by improving our techniques for nucleic acid fractionation based upon relative organism abundance as well as heuristic approaches for organism-mixture analysis. The anticipated result of this study will be a rapid, first response system capable of high-throughput microbial community analysis of a variety of sample types. Including nucleic acid isolation, data acquisition and analysis, identification(s) should be feasible in less than 1.5 hours. Rapid response to infectious disease requires that the analytical procedures employed are inherently fast. Compared to most analytical techniques, mass spectrometry is generally faster, providing both separation of complex mixtures and a fundamental measurement simultaneously. In addition to microbial identification for clinical response, diagnostics are also needed for a wide range of applications including food and water safety, bioreactor analysis and sterility assurance, and environmental microbiology. This project focuses on developing a total system for microbial identification using predictive software, novel molecular techniques, and mass spectral analysis of ribosomal RNA fragments.