DNA probe method for the detection of specific microorganisms in the soil bacterial community

• Published in: Applied and Environmental Microbiology Vol. 54; no. 3; pp. 703 - 711
• Main Authors: Holben, W.E. (Michigan State University, East Lansing, MI), Jansson, J.K, Chelm, B.K, Tiedje, J.M
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.03.1988
• Subjects: ADN; Agronomy. Soil science and plant productions; BACTERIA; Biological and medical sciences; Biotechnology; BRADYRHIZOBIUM; Bradyrhizobium japonicum; CONTROLE CONTINU; Economic plant physiology; FLORA DEL SUELO; FLORE DU SOL; Fundamental and applied biological sciences. Psychology; GENE; General Microbial Ecology; GENES; GENIE GENETIQUE; INGENIERIA GENETICA; Methods. Procedures. Technologies; Microbial engineering. Fermentation and microbial culture technology; Symbiosis (nodules, symbiotic nitrogen fixation, mycorrhiza...); VIGILANCIA; Molecular hybridization; Purification; Bradyrhizobium japonicum; Microflora; Identification; Method; DNA; Soil; Bacteria; Molecular probe; Rhizobiaceae; Detection; Method study
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.54.3.703-711.1988

We developed a protocol which yields purified bacterial DNA from the soil bacterial community. The bacteria were first dispersed and separated from soil particles in the presence of polyvinylpolypyrrolidone, which removes humic acid contaminants by adsorption to this insoluble polymer. The soil bacteria were then collected by centrifugation and lysed by using a comprehensive protocol designed to maximize disruption of the various types of bacteria present. Total bacterial DNA was purified from the cell lysate and remaining soil contaminants by using equilibrium density gradients. The isolated DNA was essentially pure as determined by UV spectral analysis, was at least 48 kilobases long, and was not subject to degradation, which indicated that there was no contaminating nuclease activity. The isolated DNA was readily digested by exogenously added restriction endonucleases and successfully analyzed by slot blot and Southern blot hybridizations. Using single-stranded, 32P-labeled DNA probes, we could detect and quantitate the presence of a specific microbial population in the natural soil community on the basis of the presence of a DNA sequence unique to that organism. The sensitivity of our methodology was sufficient to detect Bradyrhizobium japonicum at densities as low as 4.3 X 10(4) cells per g (dry weight) of soil, which corresponds to about 0.2 pg of hybridizable DNA in a 1-microgram DNA sample