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National Institute of Environmental Health Sciences

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Your Environment. Your Health.

Publication Detail

Title: Direct loop mediated isothermal amplification on filters for quantification of Dehalobacter in groundwater.

Authors: Stedtfeld, Robert D; Stedtfeld, Tiffany M; Samhan, Farag; Kanitkar, Yogendra H; Hatzinger, Paul B; Cupples, Alison M; Hashsham, Syed A

Published In J Microbiol Methods, (2016 Dec)

Abstract: Nucleic acid amplification of biomarkers is increasingly used to monitor microbial activity and assess remedial performance in contaminated aquifers. Previous studies described the use of filtration, elution, and direct isothermal amplification (i.e. no DNA extraction and purification) as a field-able means to quantify Dehalococcoides spp. in groundwater. This study expands previous work with direct loop mediated isothermal amplification (LAMP) for the detection and quantification of Dehalobacter spp. in groundwater. Experiments tested amplification of DNA with and without crude lysis and varying concentrations of humic acid. Three separate field-able methods of biomass concentration with eight aquifer samples were also tested, comparing direct LAMP with traditional DNA extraction and quantitative PCR (qPCR). A new technique was developed where filters were amplified directly within disposable Gene-Z chips. The direct filter amplification (DFA) method eliminated an elution step and provided a detection limit of 102Dehalobacter cells per 100mL. LAMP with crudely lysed Dehalobacter had a negligible effect on threshold time and sensitivity compared to lysed samples. The LAMP assay was more resilient than traditional qPCR to humic acid in sample, amplifying with up to 100mg per L of humic acid per reaction compared to 1mg per L for qPCR. Of the tested field-able concentrations methods, DFA had the lowest coefficient of variation among Dehalobacter spiked groundwater samples and lowest threshold time indicating high capture efficiency and low inhibition. While demonstrated with Dehalobacter, the DFA method can potentially be used for a number of applications requiring field-able, rapid (<60min) and highly sensitive quantification of microorganisms in environmental water samples.

PubMed ID: 27720723 Exiting the NIEHS site

MeSH Terms: Bacteriological Techniques/methods; Biomarkers/analysis; Biomass; Chloroflexi/genetics*; Chloroflexi/isolation & purification*; DNA, Bacterial/analysis; DNA, Bacterial/isolation & purification; Environmental Monitoring/instrumentation; Environmental Monitoring/methods*; Equipment Design; Filtration/methods*; Gene Dosage; Groundwater/microbiology*; Limit of Detection; Nucleic Acid Amplification Techniques/instrumentation; Nucleic Acid Amplification Techniques/methods*; RNA, Ribosomal, 16S/genetics; Real-Time Polymerase Chain Reaction/instrumentation; Real-Time Polymerase Chain Reaction/methods; Sensitivity and Specificity; Time Factors

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