Simple Absolute Quantification Method Correcting for Quantitative PCR Efficiency Variations for Microbial Community Samples

Brankatschk, Robert; Bodenhausen, Natacha; Zeyer, Josef; Bürgmann, Helmut

doi:10.1128/aem.07878-11

Cited by 187 publications

(132 citation statements)

References 53 publications

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“…Since waterborne pathogens pose a significant threat to human health and a proper assessment of microbial water quality is of urgent need, microbial water quality monitoring has undergone a tremendous transition in recent years, with the novel molecular tools beginning to offer rapid, high-throughput, sensitive, and specific detection of a wide spectrum of microbial pathogens that challenge traditional culturebased techniques. For instance, real-time quantitative PCR (qPCR), high-density microarrays, and pyrosequencing are currently used to achieve simultaneous detection and semiquantification of harmful species in environmental samples (64,65). However, in waters with no pathogenic microbe contamination, monitoring methods have traditionally been based on molecular fingerprinting approaches.…”

Section: Discussionmentioning

confidence: 99%

Molecular Fingerprinting of Cyanobacteria from River Biofilms as a Water Quality Monitoring Tool

Loza

Perona

Mateo

2013

Appl Environ Microbiol

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Benthic cyanobacterial communities from Guadarrama River (Spain) biofilms were examined using temperature gradient gel electrophoresis (TGGE), comparing the results with microscopic analyses of field-fixed samples and the genetic characterization of cultured isolates from the river. Changes in the structure and composition of cyanobacterial communities and their possible association with eutrophication in the river downstream were studied by examining complex TGGE patterns, band extraction, and subsequent sequencing of 16S rRNA gene fragments. Band profiles differed among sampling sites depending on differences in water quality. The results showed that TGGE band richness decreased in a downstream direction, and there was a clear clustering of phylotypes on the basis of their origins from different locations according to their ecological requirements. Multivariate analyses (cluster analysis and canonical correspondence analysis) corroborated these differences. Results were consistent with those obtained from microscopic observations of field-fixed samples. According to the phylogenetic analysis, morphotypes observed in natural samples were the most common phylotypes in the TGGE sequences. These phylotypes were closely related to Chamaesiphon, Aphanocapsa, Pleurocapsa, Cyanobium, Pseudanabaena, Phormidium, and Leptolyngbya. Differences in the populations in response to environmental variables, principally nutrient concentrations (dissolved inorganic nitrogen and soluble reactive phosphorus), were found. Some phylotypes were associated with low nutrient concentrations and high levels of dissolved oxygen, while other phylotypes were associated with eutrophic-hypertrophic conditions. These results support the view that once a community has been characterized and its genetic fingerprint obtained, this technique could be used for the purpose of monitoring rivers.

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Section: Discussionmentioning

confidence: 99%

Molecular Fingerprinting of Cyanobacteria from River Biofilms as a Water Quality Monitoring Tool

Loza

Perona

Mateo

2013

Appl Environ Microbiol

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“…Quantitative PCR (qPCR) assays targeting genes encoding 16S rRNA, the ␣-subunit of the dissimilatory sulfite reductase (dsrA), and the ␣-subunit of the benzylsuccinate synthase (bssA) were carried out in 10-l reaction mixtures . Gene abundances were estimated following the one-point calibration method (OPC) in order to account for possible discrepancies between PCR efficiency of the standards and the DNA extracted from the anodes and the bulk anolyte (38). From every qPCR assay, raw fluorescence data (non-baseline corrected) from individual PCRs were imported into LinRegPCR (2014.x) for optimal baseline subtraction and estimation of threshold cycle number (Cq) and efficiency (E) (39).…”

Section: Methodsmentioning

confidence: 99%

“…Cq sample (38). E standard and E sample were calculated from the arithmetic mean of triplicate reactions.…”

Section: Methodsmentioning

confidence: 99%

Anodes Stimulate Anaerobic Toluene Degradation via Sulfur Cycling in Marine Sediments

Daghio

Vaiopoulou

Patil

et al. 2016

Appl Environ Microbiol

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cHydrocarbons released during oil spills are persistent in marine sediments due to the absence of suitable electron acceptors below the oxic zone. Here, we investigated an alternative bioremediation strategy to remove toluene, a model monoaromatic hydrocarbon, using a bioanode. Bioelectrochemical reactors were inoculated with sediment collected from a hydrocarbon-contaminated marine site, and anodes were polarized at 0 mV and ؉300 mV (versus an Ag/AgCl [3 M KCl] reference electrode). The degradation of toluene was directly linked to current generation of up to 301 mA m ؊2 and 431 mA m ؊2 for the bioanodes polarized at 0 mV and ؉300 mV, respectively. Peak currents decreased over time even after periodic spiking with toluene. The monitoring of sulfate concentrations during bioelectrochemical experiments suggested that sulfur metabolism was involved in toluene degradation at bioanodes. 16S rRNA gene-based Illumina sequencing of the bulk anolyte and anode samples revealed enrichment with electrocatalytically active microorganisms, toluene degraders, and sulfate-reducing microorganisms. Quantitative PCR targeting the ␣-subunit of the dissimilatory sulfite reductase (encoded by dsrA) and the ␣-subunit of the benzylsuccinate synthase (encoded by bssA) confirmed these findings. In particular, members of the family Desulfobulbaceae were enriched concomitantly with current production and toluene degradation. Based on these observations, we propose two mechanisms for bioelectrochemical toluene degradation: (i) direct electron transfer to the anode and/or (ii) sulfide-mediated electron transfer.

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“…Reviews dealing with the advantages and limitations of qPCR have been published [9,107,135]. Accordingly, qPCR is currently the main method used for quantification of environmental samples, including the denitrifier communities.…”

Section: Quantification By Pcr Of Denitrifier Communitiesmentioning

confidence: 99%

Determination of Denitrification Genes Abundance in Environmental Samples

Correa‐Galeote¹,

Tortosa²,

Bedmar³

2013

Metagenomics

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Diversity of microorganisms involved in the biogeochemical N cycle is of fundamental interest in microbial ecology. Denitrification is a key step in the cycle by which nitrate is reduced to dinitrogen gas via the soluble nitrite and the gaseous compounds nitric oxide and nitrous oxide. The process is carried out by the sequential activity of the nitrate, nitrite, nitric oxide, and nitrous oxide reductase enzyme, respectively. The fluorescence-based quantitative real-time polymerase chain reaction (qPCR) is widely used for quantification of nucleic acids in samples obtained from numerous, diverse sources. Here, we provide a well-proven methodology for isolation of DNA from environmental samples and describe relevant experimental conditions for utilization of qPCR to assay the 16S rRNA and nar/nap, nirK/nirS, c-nor/qnor, and nos denitrification genes that encode synthesis of denitrifying enzymes. The ISO 11063 standard method and MIQUE guidelines are considered with the aim to increase experimental transparency.

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Simple Absolute Quantification Method Correcting for Quantitative PCR Efficiency Variations for Microbial Community Samples

Cited by 187 publications

References 53 publications

Molecular Fingerprinting of Cyanobacteria from River Biofilms as a Water Quality Monitoring Tool

Molecular Fingerprinting of Cyanobacteria from River Biofilms as a Water Quality Monitoring Tool

Anodes Stimulate Anaerobic Toluene Degradation via Sulfur Cycling in Marine Sediments

Determination of Denitrification Genes Abundance in Environmental Samples

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