title: Competitive touchdown PCR for estimation of Escherichia coli DNA recovery in soil DNA extraction.
abstract: 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.
authors.affiliation: Department of Soil Science and Molecular and Environmental Toxicology Center, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Rose
authors.forename: John M
authors.initials: JM
authors.lastname: Harkin
authors.forename: William J
authors.initials: WJ
authors.lastname: Hickey
mesh_terms: 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
chemical_list: D004269:DNA, Bacterial; D012987:Soil