Development and Evaluation of Micrparray-Based Whole-Genome Hybridization for Detection of Microorganisms within the Context of Environmental Applications

摘要

The detection and identification of microorganisms in natural communities is a great challenge to biologists. Microarray-based genomic technology provides a promising high-throughput alternative to traditional microbial characterization. A novel prototype microarray containing whole genomic DNA, termed community genome array (CGA), was constructed and evaluated. Microarray hybridizations at 55℃ using 50% formamide permitted the examined bacteria to be distinguished at the species level, while strain-level differentiation was obtained at hybridization temperatures of 65 or 75℃. The detection limit was estimated to be approximately 0.2 ng with genomic DNA from a single pure culture using a reduced hybridization volume (3 μL). Using mixtures of known amounts of DNA or a known number of cells from 14 or 16 different species, respectively, about 5 ng of genomic DNA or 2.5 × 10{sup}5 cells were detected underthe hybridization conditions used. In addition, strong linear relationships were observed between hybridization signal intensity and target DNA concentrations for pure cultures, a mixture of DNA templates, and a population of mixed cells (r{sup}2 = 0.95- 0.98, P < 0.01). Finally, the prototype CGA revealed differences in microbial community composition in soil, river, and marine sediments. The results suggest that CGA hybridization has potential as a specific, sensitive, and quantitative tool for detection and identification of microorganisms in environmental samples.