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DEVELOPMENT AND APPLICATION OF A RAPID, USER-FRIENDLY, AND INEXPENSIVE METHOD TO DETECT DEHALOCOCCOIDES SP. REDUCTIVE DEHALOGENASE GENES FROM GROUNDWATER
Kanitkar, Y.H., R.D. Stedtfeld, P.B. Hatzinger, S.A. Hashsham, and A.M. Cupples.
Applied Microbiology and Biotechnology 101(11):4827-4835(2017)

A novel approach for determining the abundance of Dehalococcoides sp. in groundwater samples from chlorinated solvent-contaminated sites needs only low-cost lab equipment (a bench-top centrifuge and a water bath) and performs in less time and with fewer resources compared to quantitative polymerase chain reaction (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, and 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 x 105 gene copies per L for vcrA and 1.3 x 105 gene copies per L for tceA. Both values are below the threshold recommended for effective in situ dechlorination. See more in Y.H. Kanitkar's dissertation at https://d.lib.msu.edu/etd/6667.



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