Quantification of functional genes from procaryotes in soil by PCR

Sharma, Shilpi; Radl, Viviane; Hai, Brigitte; Kloos, Karin; Fuka, Mirna Mrkonjić; Engel, Marion; Schauss, Kristina; Schloter, Michael

doi:10.1016/j.mimet.2006.10.001

Cited by 74 publications

(47 citation statements)

References 69 publications

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“…The 80 aligned sequences were used for a similar procedure to identify conserved regions. Taking into account the suitability of the amplicon for qPCR (50 to 250 bp) (35,36), only a few sites were retained. Furthermore, in order to have amplicons of a constant length for all endospore-forming species, the alignment between the annealing sites for the forward and reverse primers could not contain gaps.…”

Section: Evaluation Of Target Genes Involved In Endosporulationmentioning

confidence: 99%

Quantification of Endospore-Forming Firmicutes by Quantitative PCR with the Functional Genespo0A

Bueche

Wunderlin

Roussel-Delif

et al. 2013

Appl Environ Microbiol

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Bacterial endospores are highly specialized cellular forms that allow endospore-forming Firmicutes (EFF) to tolerate harsh environmental conditions. EFF are considered ubiquitous in natural environments, in particular, those subjected to stress conditions. In addition to natural habitats, EFF are often the cause of contamination problems in anthropogenic environments, such as industrial production plants or hospitals. It is therefore desirable to assess their prevalence in environmental and industrial fields. To this end, a high-sensitivity detection method is still needed. The aim of this study was to develop and evaluate an approach based on quantitative PCR (qPCR). For this, the suitability of functional genes specific for and common to all EFF were evaluated. Seven genes were considered, but only spo0A was retained to identify conserved regions for qPCR primer design. An approach based on multivariate analysis was developed for primer design. Two primer sets were obtained and evaluated with 16 pure cultures, including representatives of the genera Bacillus, Paenibacillus, Brevibacillus, Geobacillus, Alicyclobacillus, Sulfobacillus, Clostridium, and Desulfotomaculum, as well as with environmental samples. The primer sets developed gave a reliable quantification when tested on laboratory strains, with the exception of Sulfobacillus and Desulfotomaculum. A test using sediment samples with a diverse EFF community also gave a reliable quantification compared to 16S rRNA gene pyrosequencing. A detection limit of about 10 4 cells (or spores) per gram of initial material was calculated, indicating this method has a promising potential for the detection of EFF over a wide range of applications.

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Section: Evaluation Of Target Genes Involved In Endosporulationmentioning

confidence: 99%

Quantification of Endospore-Forming Firmicutes by Quantitative PCR with the Functional Genespo0A

Bueche

Wunderlin

Roussel-Delif

et al. 2013

Appl Environ Microbiol

View full text Add to dashboard Cite

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“…Intercalating binding dyes are non-specific and generate fluorescence when bound to dsDNA (Morrison et al 1998). Therefore, quantitation with DNA-binding dyes is usually less accurate than with sequence-specific probes because all dsDNA is detected, including primer dimmers, resulting in false positive signals (Sharma et al 2007).…”

Section: Microarraymentioning

confidence: 99%

Qualitative and quantitative methodologies for determination of airborne microorganisms at concentrated animal-feeding operations

Dungan

Leytem

2009

World J Microbiol Biotechnol

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The generation of airborne microorganisms from concentrated animal-feeding operations (CAFOs) is a concern from a human and animal health perspective. To better understand the airborne microorganisms found in these environments, a number of collection and analytical techniques have been utilized and will be discussed in this review. The most commonly used bioaerosol collection method is the liquid impingement format, which is suitable with a number of culture-based and non-culture molecularbased approaches, such as polymerase chain reaction. However, the vast majority of airborne microorganism studies conducted at CAFOs utilize culture-based analyses. Because of the limitations often associated with culturebased analyses, we focused our discussion on the application of molecular-based techniques to identify and/or quantify microorganisms, as they have promising application in bioaerosol research. The ability to rapidly characterize airborne microorganisms will help to ensure protection of public and environmental health.

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“…Previous limitations on using microorganisms as indicators of river health have largely been superseded by recent advances in molecular biology and the development of a new suite of tools and techniques that are revolutionizing environmental microbiology and microbial ecology (22,38,53). For example, culture-independent techniques are providing new insights into the phylogenetic structure of microbiological communities (23,45).…”

mentioning

confidence: 99%

Effect of Wastewater Treatment Plant Effluent on Microbial Function and Community Structure in the Sediment of a Freshwater Stream with Variable Seasonal Flow

Wakelin

Colloff

Kookana

2008

Appl Environ Microbiol

211

127

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We investigated the effects of wastewater treatment plant (WWTP) discharge on the ecology of bacterial communities in the sediment of a small, low-gradient stream in South Australia. The quantification of genes involved in the biogeochemical cycling of carbon and nitrogen was used to assess potential impacts on ecosystem functions. The effects of disturbance on bacterial community structure were assessed by PCRdenaturing gradient gel electrophoresis of 16S rRNA genes, and clone library analysis was used to phylogenetically characterize significant shifts. Significant (P < 0.05) shifts in bacterial community structures were associated with alteration of the sediment's physicochemical properties, particularly nutrient loading from the WWTP discharge. The effects were greatest at the sampling location 400 m downstream of the outfall where the stream flow is reduced. This highly affected stretch of sediment contained representatives of the gammaproteobacteria that were absent from less-disturbed sites, including Oceanospirillales and Methylococcaceae. 16S rRNA gene sequences from less-disturbed sites had representatives of the Caulobacteraceae, Sphingomonadaceae, and Nitrospirae which were not represented in samples from disturbed sediment. The diversity was lowest at the reference site; it increased with proximity to the WWTP outfall and declined toward highly disturbed (400 m downstream) sites (P < 0.05). The potential for biological transformations of N varied significantly with the stream sediment location (P < 0.05). The abundance of amoA, narG, and nifH genes increased with the distance downstream of the outfall. These processes are driven by N and C availability, as well as redox conditions. Together these data suggest cause and effect between nutrient loading into the creek, shift in bacterial communities through habitat change, and alteration of capacity for biogeochemical cycling of N.

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Quantification of functional genes from procaryotes in soil by PCR

Cited by 74 publications

References 69 publications

Quantification of Endospore-Forming Firmicutes by Quantitative PCR with the Functional Genespo0A

Quantification of Endospore-Forming Firmicutes by Quantitative PCR with the Functional Genespo0A

Qualitative and quantitative methodologies for determination of airborne microorganisms at concentrated animal-feeding operations

Effect of Wastewater Treatment Plant Effluent on Microbial Function and Community Structure in the Sediment of a Freshwater Stream with Variable Seasonal Flow

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