title: DNA extraction-free quantification of Dehalococcoides spp. in groundwater using a hand-held device.
abstract: Nucleic acid amplification of biomarkers is increasingly used to measure microbial activity and predict remedial performance in sites with trichloroethene (TCE) contamination. Field-based genetic quantification of microorganisms associated with bioremediation may help increase accuracy that is diminished through transport and processing of groundwater samples. Sterivex cartridges and a previously undescribed mechanism for eluting biomass was used to concentrate cells. DNA extraction-free loop mediated isothermal amplification (LAMP) was monitored in real-time with a point of use device (termed Gene-Z). A detection limit of 10(5) cells L(–1) was obtained, corresponding to sensitivity between 10 to 100 genomic copies per reaction for assays targeting the Dehalococcoides spp. specific 16S rRNA gene and vcrA gene, respectively. The quantity of Dehalococcoides spp. genomic copies measured from two TCE contaminated groundwater samples with conventional means of quantification including filtration, DNA extraction, purification, and qPCR was comparable to the field ready technique. Overall, this method of measuring Dehalococcoides spp. and vcrA genes in groundwater via direct amplification without intentional DNA extraction and purification is demonstrated, which may provide a more accurate mechanism of predicting remediation rates.
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, Michigan 48824, United States.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Stedtfeld
authors.forename: Tiffany M
authors.initials: TM
authors.lastname: Stedtfeld
authors.forename: Maggie
authors.initials: M
authors.lastname: Kronlein
authors.forename: Gregoire
authors.initials: G
authors.lastname: Seyrig
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Steffan
authors.forename: Alison M
authors.initials: AM
authors.lastname: Cupples
authors.forename: Syed A
authors.initials: SA
authors.lastname: Hashsham
mesh_terms: D001673:Biodegradation, Environmental; D041862:Chloroflexi / Q000235:genetics* / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000302:isolation & purification; D004784:Environmental Monitoring / Q000295:instrumentation* / Q000379:methods*; D004867:Equipment Design; D020459:Genes, rRNA; D060587:Groundwater / Q000382:microbiology*; D057230:Limit of Detection; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D014241:Trichloroethylene; D014874:Water Pollutants, Chemical
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D014874:Water Pollutants, Chemical; D014241:Trichloroethylene