title: Development and application of a rapid, user-friendly, and inexpensive method to detect Dehalococcoides sp. reductive dehalogenase genes from groundwater.
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 × 105gene copies per L for vcrA and 1.3 × 105gene copies per L for tceA. Both values are below the threshold recommended for effective in situ dechlorination.
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, A135, 1449 Engineering Research Court, East Lansing, MI, 48824, USA.
authors.forename: Yogendra H
authors.initials: YH
authors.lastname: Kanitkar
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, A135, 1449 Engineering Research Court, East Lansing, MI, 48824, USA.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Stedtfeld
authors.affiliation: CB&I Federal Services, 17 Princess Road, Lawrenceville, NJ, 08648, USA.
authors.forename: Paul B
authors.initials: PB
authors.lastname: Hatzinger
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, A135, 1449 Engineering Research Court, East Lansing, MI, 48824, USA.|Center for Microbial Ecology, Michigan State University, East Lansing, MI, USA.
authors.forename: Syed A
authors.initials: SA
authors.lastname: Hashsham
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, A135, 1449 Engineering Research Court, East Lansing, MI, 48824, USA. cupplesa@egr.msu.edu.
authors.forename: Alison M
authors.initials: AM
authors.lastname: Cupples
mesh_terms: D052160:Benzothiazoles; D001673:Biodegradation, Environmental; D018533:Biomass; D041862:Chloroflexi / Q000201:enzymology / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D003959:Diamines; D005798:Genes, Bacterial; D060587:Groundwater / Q000382:microbiology*; D054879:Halogenation / Q000235:genetics; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques / Q000191:economics / Q000379:methods; D009930:Organic Chemicals; D011804:Quinolines; D012336:RNA, Ribosomal, 16S; D014871:Water Microbiology; D014874:Water Pollutants, Chemical
chemical_list: D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012336:RNA, Ribosomal, 16S; D014874:Water Pollutants, Chemical; C098022:SYBR Green I
keywords: Dehalococcoides sp.; Direct cell amplification; LAMP; Loop-mediated isothermal amplification; Reductive dehalogenase genes; SDC-9; SYBR Green