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Results for “Letters in Applied Microbiology”

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3 recordsLinked to original sources
USGS2010

Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR

Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms. ?? 2010 The Society for Applied Microbiology.

Letters in Applied Microbiology↗
USGS2002

A rapid and efficient assay for extracting DNA from fungi

Aims: A method for the rapid extraction of fungal DNA from small quantities of tissue in a batch-processing format was investigated. Methods and Results: Tissue (< 3.0 mg) was scraped from freshly-grown fungal isolates. The tissue was suspended in buffer AP1 and subjected to seven rounds of freeze/thaw using a crushed dry ice/ethanol bath and a boiling water bath. After a 30 min boiling step, the tissue was quickly ground against the wall of the microfuge tube using a sterile pipette tip. The Qiagen DNeasy Plant Tissue Kit protocol was then used to purify the DNA for PCR/ sequencing applications. Conclusions: The method allowed batch DNA extraction from multiple fungal isolates using a simple yet rapid and reliable assay. Significance and Impact of the Study: Use of this assay will allow researchers to obtain DNA from fungi quickly for use in molecular assays that previously required specialized instrumentation, was time-consuming or was not conducive to batch processing.

Letters in Applied Microbiology↗
USGS2020

Mapping metabolic activity at single cell resolution in intact volcanic fumarole soil

Interactions among microorganisms and their mineralogical substrates govern the structure, function, and emergent properties of microbial communities. These interactions are predicated on spatial relationships, which dictate metabolite exchange and access to key substrates. To quantitatively assess links between spatial relationships and metabolic activity, this study presents a novel approach to map all organisms, the metabolically active subset, and associated mineral grains, all while maintaining spatial integrity of an environmental microbiome. We applied this method at an outgassing fumarole of Vanuatu’s Marum Crater, one of the largest point sources of several environmentally relevant gaseous compounds, including H2O, CO2, and SO2. With increasing distance from the soil-air surface and from mineral grain outer boundaries, organism abundance decreased but the proportion of metabolically active organisms often increased. These protected niches may provide more stable conditions that promote consistent metabolic activity of a streamlined community. Conversely, mineral exteriors accumulate more organisms that may cover a wider range of preferred conditions, implying that only a subset of the community will be active under any particular environmental regime. More broadly, the approach presented here allows investigators to see microbial communities “as they really are” and explore determinants of metabolic activity across a range of microbiomes.

FEMS Microbiology Letters↗