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While molecular methods are rapidly supplanting phenotypic identification of micro-organisms, the most successful approaches have employed organism-specific nucleic acid probes or primers for PCR amplification. Identification by nucleic acid hybridization therefore implies a priori knowledge (or at least suspicion) of a putative organism. Such assays are therefore limited in generality by the number of probes or primers on hand. This project describes an open or exploratory system with no such limitation which is also superior in speed to DNA sequencing. By leveraging the wealth of publicly available ribosomal RNA sequences for thousands of bacterial strains, and rapid mass spectrometry of novel, mass-modified RNA fragments, the system can identify bacterial species in complex organism mixtures and report their relative abundances. The technology is amenable to high-throughput automated analysis of over 200 samples in less than 2 hours and is compatible with any sample type from which total DNA can be isolated. Finally, the technology can be readily expanded to identification of viruses and eukaryotes as increasing sequence information on these organisms becomes available. The system will have a large beneficial impact on monitoring and protecting the biological safety of our food and water supplies.
NON-TECHNICAL SUMMARY: Rapid, exploratory diagnostics are needed for the identification of bacteria in complex food and water samples. This project focuses on development of a novel, rapid system for molecular identification of bacteria.
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APPROACH: The focus of this research project is to develop a total system for rapid microbial identification at the molecular level that eliminates the need for a large number of hybridization probes or sequencing. The system comprises five principle components: (1) rapid nucleic acid sample preparation techniques that fractionate complex samples based on the relative abundance of the organisms present (2) a simple software implementation that predicts base-specific nucleic acid fragments released from enzymatic or chemical treatment of informative RNA or DNA sequences, (3) a laboratory protocol for selectively generating base-specific fragments from a sample material, (4) matrix assisted laser desorption ionization time of flight (MALDITOF) mass spectrometry for measuring resulting fragment masses and (5) spectral processing for comparison of observed spectra to the database of predicted fragments and hence presence/absence and identification of pathogens or other organisms of interest. The anticipated result of this study will be a set of laboratory protocols, reagents, and an analytical database which together comprise a rapid, first response system that yields a confidence index regarding the organism(s) present in a food or water sample as well as their relative abundances. Including nucleic acid isolation to data acquisition and analysis, characterization of multiple samples will be feasible in less than 2 hours using widely available instrumentation. Furthermore, the system will be able to characterize complex microbial communities without the need for cloning.
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PROGRESS: 2006/05 TO 2006/12 <BR>
Rapid response to and/or forensic tracking of a biological problem in the food supply requires that the analytical procedures employed are inherently fast. Compared to most analytical techniques, mass spectrometry is one of the fastest, providing both separation of complex mixtures and a fundamental measurement simultaneously. Due to this analytical speed, mass spectrometry is also amenable to high-throughput analysis of very many samples. Finally, the widespread portability of biological mass spectrometers is very near as miniaturized spectrometers for biological applications are currently in development. A large repository of nucleic acid sequence data for over 250,000 bacteria exists in the form of the publicly available Ribosomal Database Project. 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. Our Phase I effort successfully demonstrated feasibility of using this technology to rapidly identify individual organisms from a number of sample types and even characterize complex microbial communities. Only two real alternatives to mass spectrometry exist to achieve the level of molecular, phylogenetic identification that we have demonstrated - complete gene sequencing by capillary electrophoresis and DNA microarrays. In contrast to these approaches and the required hardware, mass spectrometry is more robust, much faster, and requires less user interaction. Finally, phylogenetics based upon ribosomal RNA is considered the "gold standard" for identification of previously unencountered organisms. We have successfully identified a variety of bacterial species out of over 47,000 possible strains representing all previously sequenced bacterial taxa. This point cannot be over-emphasized - our approach provides a "universal" bacterial assay. Following in vitro transcription of universal PCR amplicons to single-stranded RNA and cleavage, fragments are rapidly characterized by mass spectrometry. All steps leading up to the rapid MALDI step are amenable to high-throughput, parallel implementation. The temporal advantage of the technology is therefore highly scaleable. For example in a 96 well format, 76 + 96/2 + 0.5 ¡Ö 124 minutes, or just over 2 hours to process 96 samples would be required. Similarly, 384 samples could be processed in under 5 hours. Finally, as we will explain, our results have shown that characterization of complex bacterial communities is possible in a single (or perhaps just a few) spectral acquisitions. It is feasible then, that analysis of a 384 well plate would not represent just identifications of 384 single bacteria, but rather characterization of 384 bacterial communities. We believe that mass spectrometry in combination with our fragmentation approach may be uniquely suited to analyze complex microbial mixtures. This realization/demonstration is one of the most exciting results to come out of the Phase I effort, and represents a possible "paradigm shift" in complex microbial community analysis.
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IMPACT: 2006/05 TO 2006/12 <BR>
The technology described here clearly has beneficial implications regarding the USDA's Strategic Plan for FY 2002-2007, specifically, 'STRATEGIC GOAL 3: ENHANCE PROTECTION AND SAFETY OF THE NATION'S AGRICULTURE AND FOOD SUPPLY'. In addition to this urgent need for microbial identification, the development of new diagnostics has broad overlap and technology transfer implications for other applications. The method will find application in a variety of markets including clinical diagnostics, biodefense and/or monitoring of large populated facilities such as airports and government buildings, municipal water supplies, and pharmaceutical manufacturing facilities. For such facilities, economics or the importance of human life can justify the purchase of a conventional mass spectrometer (currently $100-300k) for use with our products. Alternatively, we hope to offer a total platform for broad range bacterial, and eventually, fungal and viral, identification.