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

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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.