期刊
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
卷 101, 期 11, 页码 4827-4835出版社
SPRINGER
DOI: 10.1007/s00253-017-8203-y
关键词
Direct cell amplification; Loop-mediated isothermal amplification; LAMP; SYBR Green; SDC-9; Dehalococcoides sp.; Reductive dehalogenase genes
资金
- United States Department of Defense SERDP grant [ER-2309, W912HQ-13-C-0071]
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 x 10(5) gene copies per L for vcrA and 1.3 x 10(5) gene copies per L for tceA. Both values are below the threshold recommended for effective in situ dechlorination.
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