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 × 10 gene copies per L for vcrA and 1.3 × 10 gene copies per L for tceA. Both values are below the threshold recommended for effective in situ dechlorination.

Rapid detection of cytochrome cd1-containing nitrite reductase encoding gene nirS of denitrifying bacteria with loop-mediated isothermal amplification assay

“…LAMP has the potential to circumvent these problems due to a reduced dependence on pretreatment of samples and the ability to conduct LAMP under isothermal condition 18,22 . In particular, the efficacy of cell-template based LAMP assay considerably enhances its application in point-of-care settings 26,31,32 . For example, we successfully detected nirS gene of P. aeruginosa cells over a range of 3.36 × 10 2 -3.36 × 10 8 CFU/reaction.…”

“…2b, cell-template based LAMP assays were carried out using the same method described for the nirS gene, but with P. aeruginosa cells as template rather than extracted genomic DNA. P. aeruginosa cells were obtained using the methods described by Kanitkar et al 31 Briefly, after the concentration of bacterial cells was quantified using the plate counting method described above, 10 mL of culture medium was centrifuged at 13,000g for 15 min to obtain a biomass pellet. The biomass pellet was then suspended in an appropriate volume of water and 2 μL of the bacterial suspension was immediately used as amplification template.…”

Abundance of Chlorinated Solvent and 1,4-Dioxane Degrading Microorganisms at Five Chlorinated Solvent Contaminated Sites Determined via Shotgun Sequencing

“…Additional site information, when available, has also been provided (e.g., plume maps, plot layouts, concentration data over time) for each site (SI Figures S1–S15). DNA was extracted (collection on a filter, bead-beating and chemical lysis) from groundwater and mixed culture (SDC-9) samples using the PowerWater DNA isolation kit (Mo Bio Laboratories, a Qiagen Company) and previously described methods. , …”

“…With the expansion of this remedial practice over the past decade, the number of sites in the US now numbers well over 2300, and bioaugmentation has been performed in at least 11 other countries ( P Hatzinger, Pers comm ). Following bioaugmentation, remediation professionals commonly monitor D. mccartyi populations, typically targeting reductive dehalogenase (RDase) genes such as vcrA − using quantitative PCR (qPCR) on nucleic acids extracted from groundwater. − …”

Metabolome patterns identify active dechlorination in bioaugmentation consortium SDC-9™

“…Molecular biological tools (MBTs) targeting biomarker genes, transcripts, and/or proteins of organohalide-respiring bacteria are employed for site assessment and bioremediation monitoring ( Amos et al, 2008 ; Matturro and Rossetti, 2015 ; Kanitkar et al, 2017 ). Quantitative PCR (qPCR) targeting of Dehalococcoides mccartyi ( Dhc ) biomarker genes serves as a robust MBT in bioremediation monitoring regimes, and a Dhc population exceeding cell abundances of 10 7 l −1 is thought to be sufficiently large to reduce the contaminant concentrations over time periods acceptable for most bioremediation projects ( Lu et al, 2006 ).…”