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

描述（由申请人提供）：世界卫生组织估计，每小时有1,600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%，是美国第三大死亡原因。除了用于临床反应的微生物鉴定外，诊断还需要广泛的应用，包括食品和水安全，生物反应器分析，制药工业的无菌保证，环境微生物学和国土安全。本研究的重点是开发和表征一个微生物鉴定平台，使用非常快速的MALDI-TOF质谱的修饰核糖体RNA片段。通过产生针对质谱表征优化的碱基特异性RNA片段，我们已经表明，“生物体纯”样品，如单菌落和克隆，可以在约1000个细胞中鉴定。1.5小时该平台是“开放的”，因为它可以鉴定先前已经测序的任何细菌生物体，而无需序列特异性核酸探针的先验设计。此外，该测定比核酸测序更快速，并且适合于鉴定生物混合物中的生物体并确定其相对丰度。最后，病原体可以在微生物或宿主核酸的不感兴趣的背景下鉴定。我们计划建立在我们有前途的系统，通过改进我们的技术，核酸分馏的基础上相对生物丰度，启发式方法，和克隆PCR的有机体混合物分析的一般细菌鉴定。这项研究的预期结果将是一个快速的，第一反应系统能够高通量微生物群落分析的各种样品类型。包括核酸的分离、数据采集和分析。384次分析应在不到5小时内可行。公共卫生相关性：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，质谱法通常更快，可以同时分离复杂的混合物和基本测量。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。该项目的重点是开发一个使用预测软件，新的分子技术和核糖体RNA片段的质谱分析进行微生物鉴定的总平台系统。

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 system for general bacterial identification by improving our techniques for nucleic acid fractionation based upon relative organism abundance, heuristic approaches, and clonal PCR 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, ca. 384 analyses should be feasible in less about 5 hours. PUBLIC HEALTH RELEVANCE: 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 platform system for microbial identification using predictive software, novel molecular techniques, and mass spectral analysis of ribosomal RNA fragments.