Impacts of Sampling and Handling Procedures on DNA- and RNA-based Microbial Characterization and Quantification of Groundwater and Saturated Soil.

摘要

The objective is to accurately evaluate the performance of groundwater bioremediation processes, methods that can quantify the populations and the in-situ activity of relevant groups of microorganisms are needed. Molecular biological techniques that rely on DNA extraction and PCR amplification are widely used to determine microbial community structure and the concentrations of specific organisms or genes in environmental samples. A study was conducted to determine an optimal sample handling and processing strategy that will yield accurate results for real-time PCR and Terminal Restriction Fragment Length Polymorphism in soil and aquifer sediments. Technical Approach The effect of DNA extraction methods and sample mass on the observed microbial community, the detection of the 16S rRNA genes for Bacteria and Dehalococcoides spp., and the tceA gene that codes for trichloroethylene (TCE) reductase found in some Dehalococcoides strains, was assessed. In addition, the effects of storage temperature, time, and condition on quantitative Dehalococcoides 16S rRNA and tceA genes, and two VC reductase functional genes (vcrA and bvc) in both groundwater and soil samples were investigated. Homogenized aquifer sediment was inoculated with a mixed Dehalococcoides consortium (KB-1) and an E. coli clone containing the tceA gene. DNA was extracted from three sample masses using three commercial DNA extraction methods. A new laboratory method for DNA extraction was also developed. In addition, two indirect methods that use cell separation steps prior to DNA extraction were assessed. Quantitative-PCR was used to measure concentrations of the 16S rRNA gene of Dehalococcoides spp and the tceA gene. T-RFLP was used to characterize the bacterial and archaeal community profiles. Results Sample mass did not affect Q-PCR results for DNA extracted using the lab method but gave inconsistent results when the kits were used.