Competitive touchdown PCR for estimation of Escherichia coli DNA recovery in soil DNA extraction.
Competitive approaches have shown promise for overcoming some of the difficulties in the use of PCR for assessment of specific bacterial species in soil. A competitive touchdown PCR (cTD-PCR) protocol specific for the rrsB gene of Escherichia coli was developed for tracking the organism in environments impacted by human wastes. Regression of product ratios from co-amplification of varying amounts of analyte and competitor DNA templates was linear. To test the robustness of the method, reactions were titrated with an extract of sterilized soil; no significant effect was detected. The cTD-PCR was used to assay recovery of E. coli DNA from soil. Stock DNA was spiked onto two sterilized soils during extraction, and the purified extracts were analyzed by cTD-PCR. Recovery of DNA spiked at a rate of 180 ng g(-1) was 34+/-7% (mean+/-S.D.) for an agricultural silt loam. DNA spiked at 1.8 pg g(-1) was recovered at a mean rate of 6.1+/-1.3%. DNA in these extracts was not directly quantifiable by image analysis. The cTD-PCR method provides a useful means of quantifying small amounts of E. coli DNA, and could be modified for other specific targets in a mixture of DNA from a variety of organisms.
Rose
Peter
P
Department of Soil Science and Molecular and Environmental Toxicology Center, University of Wisconsin-Madison, Madison, WI 53706, USA.
Harkin
John M
JM
Hickey
William J
WJ
D015139:Blotting, Southern; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D007091:Image Processing, Computer-Assisted; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012987:Soil / Q000032:analysis*; D012988:Soil Microbiology
D004269:DNA, Bacterial; D012987:Soil