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Microbial DNA Analysis in the Field Using a Biological Extraction Field Kit and a Field qPCR Unit
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DNA extraction-free quantification of Dehalococcoides spp. in groundwater using a hand-held device
Robert D Stedtfeld1, Tiffany M Stedtfeld, Maggie Kronlein
1Department of Civil and Environmental Engineering, Michigan State University, East Lansing, Michigan 48824, United States.
Environmental Science & Technology
|November 1, 2014
Summary
This study introduces a rapid, field-based method for quantifying microbial biomarkers in trichloroethene (TCE) contaminated groundwater. The DNA extraction-free approach accurately predicts bioremediation rates by directly amplifying key genes.
Area of Science:
- Environmental microbiology
- Bioremediation science
- Molecular diagnostics
Background:
- Nucleic acid amplification of biomarkers is crucial for assessing microbial activity and predicting bioremediation success in trichloroethene (TCE) contaminated sites.
- Current methods involving sample transport and processing can reduce the accuracy of microbial quantification.
- Field-based genetic quantification offers a potential solution to improve accuracy.
Purpose of the Study:
- To develop and validate a field-ready method for direct genetic quantification of microorganisms involved in TCE bioremediation.
- To assess the accuracy and sensitivity of a DNA extraction-free loop-mediated isothermal amplification (LAMP) technique for microbial biomarker detection.
- To compare the performance of the field-based method with conventional laboratory techniques.
Main Methods:
- Utilized Sterivex cartridges for cell concentration and a novel elution mechanism.
- Employed DNA extraction-free loop-mediated isothermal amplification (LAMP) monitored in real-time using a point-of-use device (Gene-Z).
- Targeted Dehalococcoides spp. specific 16S rRNA and vcrA genes, achieving a detection limit of 10(5) cells L(–1).
Main Results:
- The field-ready LAMP assay demonstrated comparable quantification of Dehalococcoides spp. and vcrA genes to conventional methods (filtration, DNA extraction, qPCR).
- A detection limit of 10(5) cells L(–1) was achieved, with sensitivity between 10 to 100 genomic copies per reaction.
- The direct amplification method without DNA extraction provided comparable results to traditional, more complex laboratory procedures.
Conclusions:
- A field-deployable method for direct amplification of microbial biomarkers (Dehalococcoides spp. and vcrA genes) in TCE-contaminated groundwater was successfully demonstrated.
- This DNA extraction-free approach offers a more accurate and efficient mechanism for predicting bioremediation rates.
- The Gene-Z device and LAMP assay provide a valuable tool for real-time monitoring of bioremediation processes.
