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Appl Environ Microbiol, July 1998, p. 2463-2472, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Small-Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil

Cheryl R. Kuske,1,* Kaysie L. Banton,1 Dante L. Adorada,1 Peter C. Stark,2 Karen K. Hill,1 and Paul J. Jackson1

Life Sciences Division1 and Chemical Science and Technology Division,2 Los Alamos National Laboratory, Los Alamos, New Mexico 87545

Received 25 August 1997/Accepted 1 May 1998

Efficient, nonselective methods to obtain DNA from the environment are needed for rapid and thorough analysis of introduced microorganisms in environmental samples and for analysis of microbial community diversity in soil. A small-scale procedure to rapidly extract and purify DNA from soils was developed for in-the-field use. Amounts of DNA released from bacterial vegetative cells, bacterial endospores, and fungal conidia were compared by using hot-detergent treatment, freeze-thaw cycles, and bead mill homogenization. Combining a hot-detergent treatment with bead mill homogenization gave the highest DNA yields from all three microbial cell types and provided DNA from the broadest range of microbial groups in a natural soil community. Only the bead mill homogenization step was effective for DNA extraction from Bacillus globigii (B. subtilis subsp. niger) endospores or Fusarium moniliforme conidia. The hot-detergent-bead mill procedure was simplified and miniaturized. By using this procedure and small-scale, field-adapted purification and quantification procedures, DNA was prepared from four different soils seeded with Pseudomonas putida cells or B. globigii spores. In a New Mexico soil, seeded bacterial targets were detected with the same sensitivity as when assaying pure bacterial DNA (2 to 20 target gene copies in a PCR mixture). The detection limit of P. putida cells and B. globigii spores in different soils was affected by the amount of background DNA in the soil samples, the physical condition of the DNA, and the amount of DNA template used in the PCR.

* Corresponding author. Mailing address: M888, Environmental Molecular Biology Group, Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545. Phone: (505) 665-4800. Fax: (505) 665-6894. E-mail: kuske{at}lanl.gov.

Appl Environ Microbiol, July 1998, p. 2463-2472, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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