title: Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9.
abstract: Real-time quantitative PCR (qPCR) protocols specific to the reductive dehalogenase (RDase) genes vcrA, bvcA, and tceA are commonly used to quantify Dehalococcoides spp. in groundwater from chlorinated solvent-contaminated sites. In this study, loop-mediated isothermal amplification (LAMP) was developed as an alternative approach for the quantification of these genes. LAMP does not require a real-time thermal cycler (i.e., amplification is isothermal), allowing the method to be performed using less-expensive and potentially field-deployable detection devices. Six LAMP primers were designed for each of three RDase genes (vcrA, bvcA, and tceA) using Primer Explorer V4. The LAMP assays were compared to conventional qPCR approaches using plasmid standards, two commercially available bioaugmentation cultures, KB-1 and SDC-9 (both contain Dehalococcoides species). DNA was extracted over a growth cycle from KB-1 and SDC-9 cultures amended with trichloroethene and vinyl chloride, respectively. All three genes were quantified for KB-1, whereas only vcrA was quantified for SDC-9. A comparison of LAMP and qPCR using standard plasmids indicated that quantification results were similar over a large range of gene concentrations. In addition, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP was comparable to that of qPCR. The developed LAMP assays for vcrA and tceA genes were validated by comparing quantification on the Gene-Z handheld platform and a real-time thermal cycler using DNA isolated from eight groundwater samples obtained from an SDC-9-bioaugmented site (Tulsa, OK). These assays will be particularly useful at sites subject to bioaugmentation with these two commonly used Dehalococcoides species-containing cultures.
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Yogendra H
authors.initials: YH
authors.lastname: Kanitkar
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Stedtfeld
authors.affiliation: Biotechnology Development and Applications Group, CB&I Federal Services, LLC, Lawrenceville, New Jersey, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Steffan
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, Michigan, USA.|Center for Microbial Ecology, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Syed A
authors.initials: SA
authors.lastname: Hashsham
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, Michigan, USA cupplesa@egr.msu.edu.
authors.forename: Alison M
authors.initials: AM
authors.lastname: Cupples
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D015415:Biomarkers / Q000032:analysis*; D041862:Chloroflexi / Q000235:genetics / Q000302:isolation & purification*; D018628:Gene Dosage; D005798:Genes, Bacterial; D060587:Groundwater / Q000382:microbiology*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D013997:Time Factors
chemical_list: D015415:Biomarkers