Quantifying the Presence and Activity of Aerobic, Vinyl Chloride-Degrading Microorganisms in Dilute Groundwater Plumes by Using Real-Time PCR
Final rept. 20 Apr 2009-5 Apr 2013
IOWA UNIV IOWA CITY
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Vinyl chloride VC, a known human carcinogen with a USEPA maximum contaminant level of 2 ppb, is a significant contaminant of concern present as dilute groundwater plumes at many DoD sites. The objective of this project is to develop quantitative real-time PCR qPCR and reverse-transcription qPCR RT-qPCR techniques to estimate the abundance and activity of aerobic, VC-oxidizing bacteria including methanotrophs and etheneotrophsVCassimilating bacteria in groundwater. Two functional genes known to participate in VC oxidation in aerobic, ethene- and VC-assimilating bacteria the alkene monooxygenase subunit gene etnC and the epoxyalkaneCoM transferase gene etnE were targeted for quantification. Using known etnC and etnE sequences from isolates, enrichment cultures and environmental samples, two sets of degenerate qPCR primers were developed for each functional gene. After extensive testing of primer specificity, we developed a SYBR green-based qPCR method for quantification of etnC and etnE. The qPCR method for etheneotrophs was extended to incorporate mRNA analysis i.e. RT-qPCR, which entailed selecting appropriate reference nucleic acids ref mRNA or ref DNA and adding known amounts of these reference material into samples following the RNA and DNA extraction steps, respectively. Following conversion of RNA to cDNA by reverse transcription RT, the abundance of ref nucleic acids was quantified alongside the etnC and etnE genes on the same qPCR plate. This facilitated calculation of the percent ref nucleic acid recovery The ref nucleic acid recovery allows assessment of RNA and DNA losses in the sample during several steps in the protocol e.g. during the RT step. The qPCR method for etheneotrophs was successfully applied to 9 different samples from three different VC-contaminated sites, in some cases over a 3 year period.
- Genetic Engineering and Molecular Biology
- Inorganic Chemistry
- Water Pollution and Control