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

Abstract

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 moniliformeconidia. 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 orB. 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.

Diverse microbial communities inhabiting ferromanganese deposits in Lechuguilla and Spider Caves

“…Nucleic acids were extracted and purified from two 0.5 g aliquots of sample or from cells extracted from enrichments with sterile syringes, using the bead-mill homogenization procedure described by Kuske et al (1998). After bead-mill disruption and centrifugation, the supernatant was transferred and the bead pellet was washed once with 1 ml of TE buffer [10 mM Tris (pH 8.0), 1 mM EDTA], rehomogenized for 5 s and centrifuged again.…”

“…Nucleic acids were precipitated from the solution by using 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ethanol, incubation on ice and centrifugation for 30 min at 12 000 g. Precipitated nucleic acids were suspended in TE. DNA was purified away from contaminants by using Sephadex G-200 spin columns equilibrated in TE (Kuske et al, 1998). The clear column eluate containing DNA was precipitated and suspended in TE buffer.…”

Sampling strategy in molecular microbial ecology: influence of soil sample size on DNA fingerprinting analysis of fungal and bacterial communities

“…1). These DNA yields were in the same order of magnitude of those classically obtained in different soil environments with various protocols (Zhou et al ., 1996;Kuske et al ., 1998;Frostegard et al ., 1999). For Auxonne and Châteaurenard soils, DNA yields were positively correlated with the size of the samples (R of 0.90 and 0.88, respectively), this trend being more marked for samples > 1 g. This observation could be related to a technical bias limiting DNA recovery in samples smaller than 1 g such as a lower lysis efficiency.…”

Efficient recovery of environmental DNA for expression cloning by indirect extraction methods

“…To emphasize the e¡ects of the di¡erent methods, all protocols comprised the same basic procedure for cell lysis (lysozyme, proteinase K, and hot SDS treatment) followed by chloroform extraction and isopropanol precipitation of the released DNA. lyse a large number of microorganisms [37,38], soft direct lysis outperformed the harsh treatment by recovering up to two times more DNA from most environmental samples. Whereas Jacobsen and Rasmussen [21] found about equal DNA yields from a seeded soil by both indirect methods, our results show two-to eight-fold higher DNA recoveries with blending.…”