Development and evaluation of microarray-based whole-genome hybridization for detection of microorganisms within the context of environmental applications.
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 degrees C 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 degrees C. 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 microL). 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 x 10(5) cells were detected under the 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 (r2 = 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.
Wu
Liyou
L
Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA.
Thompson
Dorothea K
DK
Liu
Xueduan
X
Fields
Matthew W
MW
Bagwell
Christopher E
CE
Tiedje
James M
JM
Zhou
Jizhong
J
D001419:Bacteria / Q000235:genetics; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis*; D004784:Environmental Monitoring / Q000379:methods; D016678:Genome; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D012988:Soil Microbiology; D014871:Water Microbiology
D015342:DNA Probes; D004269:DNA, Bacterial