Rose Peter, Harkin John M, Hickey William J
Department of Soil Science and Molecular and Environmental Toxicology Center, University of Wisconsin-Madison, Madison, WI 53706, USA.
J Microbiol Methods. 2003 Jan;52(1):29-38. doi: 10.1016/s0167-7012(02)00131-8.
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.
竞争法已显示出有望克服在使用聚合酶链反应（PCR）评估土壤中特定细菌种类时遇到的一些困难。针对大肠杆菌rrsB基因开发了一种竞争性降落PCR（cTD-PCR）方案，用于追踪受人类粪便影响环境中的该生物体。不同量分析物和竞争DNA模板共扩增产物比例的回归呈线性。为测试该方法的稳健性，用灭菌土壤提取物对反应进行滴定；未检测到显著影响。cTD-PCR用于测定从土壤中回收的大肠杆菌DNA。在提取过程中将储备DNA添加到两种灭菌土壤中，并用cTD-PCR分析纯化提取物。对于农业粉质壤土，以180 ng g(-1)的速率添加的DNA回收率为34±7%（平均值±标准差）。以1.8 pg g(-1)添加的DNA平均回收率为6.1±1.3%。这些提取物中的DNA无法通过图像分析直接定量。cTD-PCR方法为定量少量大肠杆菌DNA提供了一种有用手段，并且可以针对来自多种生物体的DNA混合物中的其他特定靶标进行修改。
