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

通过核糖体 RNA 的 MALDI-TOF 质谱法快速鉴定微生物

• 批准号: 8059006
• 负责人: George W Jackson
• 金额: $ 20.89万
• 依托单位: BIOTEX, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-19 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8059006
• 关键词: 5 year old; Accounting; Address; Animals; Antibiotics; Area; Automation; Bacillus anthracis; Bacteria; Base Sequence; Biological; Biological Assay; Bioreactors; Blood; Brucella; Categories; Cause of Death; Chemical Agents; Child; Clinic; Clinical; Cloning; Collaborations; Communicable Diseases; Communities; Complex; Complex Mixtures; Computer software; DNA; DNA Microarray Chip; Data; Databases; Development; Devices; Diagnostic; Disease; Drug Industry; Emulsions; Engineering; Environment; Environmental Microbiology; Evaluation; Eye; Eye Infections; Family Picornaviridae; Filtration; Flavivirus; Food; Foot-and-Mouth Disease; Fractionation; Genes; Genotype; Genus Mycobacterium; Goals; Heating; Hour; Infection; Informatics; Internet; Laboratories; MALDI-TOF Mass Spectrometry; Marketing; Mass Spectrum Analysis; Measurement; Measures; Methods; Mind; Molecular; National Institute of Allergy and Infectious Disease; Nucleic Acid Probes; Nucleic Acids; Nucleic acid sequencing; Oligoribonucleotides; Oral; Organism; Pattern; Phase; Procedures; Process; Protocols documentation; RNA; RNA Sequences; Refractory; Relative (related person); Research; Research Infrastructure; Research Personnel; Research Project Grants; Ribosomal RNA; Rickettsia; Safety; Sampling; Security; Small Business Innovation Research Grant; Sorting - Cell Movement; Speed; Sterility; System; Techniques; Technology; Texas; Time; Toxin; United States; United States National Institutes of Health; Universities; Water; Work; World Health Organization; Yersinia pestis; base; biosecurity; clinically relevant; commercial application; data acquisition; data structure; design; eye center; genotyping technology; heuristics; improved; instrument; killings; mass spectrometer; method development; microbial; microbial community; miniaturize; mortality; novel; operation; pathogen; public health relevance; rapid technique; response

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.

描述（由申请人提供）：世界卫生组织估计，每小时有超过 1,600 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%，是美国的第三大死因。除了临床反应的微生物鉴定之外，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。本研究的重点是使用修饰核糖体 RNA 片段的快速 MALDI-TOF 质谱法开发和表征微生物鉴定平台。通过生成针对质谱表征进行优化的碱基特异性 RNA 片段，我们已经证明，可以在约 100% 的时间内鉴定出“有机体纯”样品，例如单菌落和克隆。 1.5小时。该平台是“开放的”，因为它可以识别先前已测序的任何细菌生物体，而无需先验设计序列特异性核酸探针。此外，该测定比核酸测序更快，并且适合鉴定生物混合物中的生物体并确定其相对丰度。最后，可以在共生生物或宿主核酸的无趣背景下识别病原体。我们计划通过改进基于相对生物体丰度、启发式方法和用于生物体混合物分析的克隆 PCR 的核酸分级分离技术，建立有前途的一般细菌鉴定系统。这项研究的预期结果将是一个快速的第一响应系统，能够对各种样本类型进行高通量微生物群落分析。包括核酸分离、数据采集和分析等。第384章 分析应该在不到5个小时的时间内可行。公共卫生相关性：对传染病的快速反应要求所采用的分析程序本质上是快速的。与大多数分析技术相比，质谱分析通常速度更快，可以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之外，广泛的应用也需要诊断，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。该项目的重点是使用预测软件、新颖的分子技术和核糖体 RNA 片段的质谱分析来开发微生物鉴定的整体平台系统。

项目成果

Detection and typing of viruses using broadly sensitive cocktail-PCR and mass spectrometric cataloging: demonstration with dengue virus.

使用广泛敏感的鸡尾酒 PCR 和质谱编目对病毒进行检测和分型：用登革热病毒进行演示。

• 发表时间: 2012-07
• 作者: Gijavanekar, Charul;Drabek, Rafal;Soni, Mithil;Jackson, George W;Strych, Ulrich;Fox, George E;Fofanov, Yuriy;Willson, Richard C
• 通讯作者: Willson, Richard C