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

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 102 Dehalobacter cells per 100 mL. 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 100 mg per L of humic acid per reaction compared to 1 mg 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 (<60 min) and highly sensitive quantification of microorganisms in environmental water samples.

Quantification of microRNAs directly from body fluids using a base-stacking isothermal amplification method in a point-of-care device

“…This allows greater distinction between levels of microRNA. The coefficient of variation among all tested samples was also low (mean CV = 2.6%), which is near or below the CV observed in other studies using isothermal amplification following DNA extraction (Bosward et al 2016; Brotons et al 2016) or directly from groundwater samples (Stedtfeld et al 2016). …”

Isothermal assay targeting class 1 integrase gene for environmental surveillance of antibiotic resistance markers

“…Gene-Z chips were fabricated as previously described (Stedtfeld et al, 2016a) and loaded with primers by trained staff at MSU. Fabrication of Gene-Z chips included cutting channels and wells into 1.59 mm thick black acrylic sheets (24112-07, Inventables) with a CO 2 laser, cleaning as previously described, and enclosing one side using clear optical film (MicroAmp, Applied Biosystems).…”

“…Next, primers were dehydrated in the Gene-Z chip, enclosed with PCR tape, and stored at −20 °C until use. LAMP assays dehydrated on the Gene-Z chip included the intI1 gene primer, universal bacteria 16S rRNA gene primer, and a positive control assay targeting the luciferase gene (Hatt et al, 2013; Stedtfeld et al, 2016a). Chips were designed so that four samples could be loaded per chip.…”

“…The application sorts, plots, and emails raw data. Data was processed further using excel as previously described to generate threshold time (Tt) akin to threshold cycle with qPCR (Stedtfeld et al, 2016a). Gene quantities from the intI1 gene and universal 16S rRNA gene LAMP assays were estimated based on standard curves and normalized to 16S rRNA gene copy number to generate relative abundance (Stalder et al, 2012).…”

“…However, more rapid and simplified measurement techniques may increase broader monitoring/surveillance of intI1 as a marker of ARG abundance or anthropogenic activity. Isothermal amplification, such as loop mediated isothermal amplification (LAMP) with Bst polymerase provides a more simplistic approach because it allows amplification with minimal sample processing requirements (Williams et al, 2017) and is less influenced by PCR inhibition (Koloren et al, 2011; Stedtfeld et al, 2016a). Furthermore, constant temperature (Notomi et al, 2000) and high amplicon yields (Mori et al, 2001), allows quantification of abundance with relatively simpler devices (e.g.…”