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Development and application of a rapid, user-friendly, and inexpensive method to detect Dehalococcoides sp. reductive dehalogenase genes from groundwater

  • Environmental biotechnology
  • Published:
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Abstract

TaqMan probe-based quantitative polymerase chain reaction (qPCR) specific to the biomarker reductive dehalogenase (RDase) genes is a widely accepted molecular biological tool (MBT) for determining the abundance of Dehalococcoides sp. in groundwater samples from chlorinated solvent-contaminated sites. However, there are significant costs associated with this MBT. In this study, we describe an approach that requires only low-cost laboratory equipment (a bench top centrifuge and a water bath) and requires less time and resources compared to qPCR. The method involves the concentration of biomass from groundwater, without DNA extraction, and loop-mediated isothermal amplification (LAMP) of the cell templates. The amplification products are detected by a simple visual color change (orange/green). The detection limits of the assay were determined using groundwater from a contaminated site. In addition, the assay was tested with groundwater from three additional contaminated sites. The final approach to detect RDase genes, without DNA extraction or a thermal cycler, was successful to 1.8 × 105 gene copies per L for vcrA and 1.3 × 105 gene copies per L for tceA. Both values are below the threshold recommended for effective in situ dechlorination.

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Acknowledgements

We would like to thank Simon Vainberg, Sheryl Streger, Robert E. Mayer, Michael Martinez, David Lippincott, and Phil Dennis for providing the groundwater and culture samples. We also thank Dr. Frank Löffler (UTK) for providing the tceA plasmid standard. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the US Government. Views, opinions, and/or findings contained in this report are those of the authors and should not be construed as an official Department of Defense position or decision unless designated by other official documentation.

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Authors and Affiliations

  1. Department of Civil and Environmental Engineering, Michigan State University, A135, 1449 Engineering Research Court, East Lansing, MI, 48824, USA

    Yogendra H. Kanitkar, Robert D. Stedtfeld, Syed A. Hashsham & Alison M. Cupples

  2. CB&I Federal Services, 17 Princess Road, Lawrenceville, NJ, 08648, USA

    Paul B. Hatzinger

  3. Center for Microbial Ecology, Michigan State University, East Lansing, MI, USA

    Syed A. Hashsham

Authors
  1. Yogendra H. Kanitkar
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  2. Robert D. Stedtfeld
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  3. Paul B. Hatzinger
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  4. Syed A. Hashsham
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  5. Alison M. Cupples
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Corresponding author

Correspondence to Alison M. Cupples.

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This research was funded by the United States Department of Defense SERDP grant ER-2309 (Contract W912HQ-13-C-0071). This article does not contain any studies with human participants or animals performed by any of the authors.

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The authors declare that they have no conflict of interest.

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Kanitkar, Y.H., Stedtfeld, R.D., Hatzinger, P.B. et al. Development and application of a rapid, user-friendly, and inexpensive method to detect Dehalococcoides sp. reductive dehalogenase genes from groundwater. Appl Microbiol Biotechnol 101, 4827–4835 (2017). https://doi.org/10.1007/s00253-017-8203-y

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  • DOI: https://doi.org/10.1007/s00253-017-8203-y

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