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，制药行业的无菌保证，环境微生物学和国土安全。本研究的重点是利用非常快速的MALDI-TOF质谱法对修饰的核糖体RNA片段进行微生物鉴定平台的开发和表征。通过生成针对质谱表征优化的碱基特异性RNA片段，我们已经证明，“生物纯”样品（如单个菌落和克隆）可以在大约1.5小时内进行鉴定。该平台是“开放的”，因为它可以识别任何细菌有机体，而无需预先设计序列特异性核酸探针。此外，该分析比核酸测序更快，适用于生物混合物中的生物鉴定和相对丰度的测定。最后，病原体可以在无趣的共生生物或宿主核酸背景下被识别出来。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，在我们有前途的普通细菌鉴定原型系统的基础上建立。本研究的预期结果将是一个能够对各种样品类型进行高通量微生物群落分析的快速，第一反应系统。包括核酸分离、数据采集和分析，鉴定应在1.5小时内可行。

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.