scholarly journals An Improved Method for Soil DNA Extraction to Study the Microbial Assortment within Rhizospheric Region

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Faria Fatima ◽  
Neelam Pathak ◽  
Smita Rastogi Verma
Keyword(s):  
Microbial Community ◽  
Soil Sample ◽  
Dna Extraction ◽  
Single Type ◽  
Soil Dna ◽  
Recovery Method ◽  
Dna Recovery ◽  
Soil Microbial ◽  
Soil Dna Extraction ◽  
Indigenous Microbial Community

The need for identification of soil microbial community mainly depends on direct extraction of DNA from soil, a multifaceted environment that is a major pool for microbial genetic diversity. The soil DNA extraction procedures usually suffer from two major problems, namely, inappropriate rupturing of cells and contamination with humic substances. In the present study, five protocols for single type of rhizospheric soil were investigated and their comparison indicated that the inclusion of 120 mM phosphate buffered saline (PBS) for washing and mannitol in the lysis buffer allowed the processing of soil sample in minimal time with no specific equipment requirement. Furthermore, DNA purity and yield were also improved, which allowed the exploitation of genetic potential of soil microbes within soil sample thereby facilitating the amplification of metagenomic DNA. The effectiveness of methods was analyzed using random amplification of polymorphic DNA. The banding patterns revealed that both the abundance and the composition of indigenous microbial community depend on the DNA recovery method.

scholarly journals DNA Extraction from Soils: Old Bias for New Microbial Diversity Analysis Methods

2001 ◽  
Vol 67 (5) ◽  
pp. 2354-2359 ◽  
Author(s):  
F. Martin-Laurent ◽  
L. Philippot ◽  
S. Hallet ◽  
R. Chaussod ◽  
J. C. Germon ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Ribosomal Dna ◽  
Dna Analysis ◽  
Restriction Analysis ◽  
Farmyard Manure ◽  
Soil Biodiversity ◽  
Soil Dna ◽  
Recovery Method ◽  
Dna Recovery ◽  
The Impact

ABSTRACT The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years.

scholarly journals Molecular biomass and MetaTaxogenomic assessment of soil microbial communities as influenced by soil DNA extraction procedure

2011 ◽  
Vol 5 (1) ◽  
pp. 135-141 ◽  
Author(s):  
Sébastien Terrat ◽  
Richard Christen ◽  
Samuel Dequiedt ◽  
Mélanie Lelièvre ◽  
Virginie Nowak ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Dna Extraction ◽  
Extraction Procedure ◽  
Soil Microbial Communities ◽  
Soil Dna ◽  
Soil Microbial ◽  
Soil Dna Extraction

Soil microbial diversity: an ISO standard for soil DNA extraction

2010 ◽  
Vol 10 (7) ◽  
pp. 1344-1345 ◽  
Author(s):  
Laurent Philippot ◽  
Cristina Abbate ◽  
Antonio Bispo ◽  
Thierry Chesnot ◽  
Sara Hallin ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
Dna Extraction ◽  
Soil Microbial Diversity ◽  
Soil Dna ◽  
Iso Standard ◽  
Soil Microbial ◽  
Soil Dna Extraction

scholarly journals Validating DNA Extraction Protocols for Bentonite Clay

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Katja Engel ◽  
Sara Coyotzi ◽  
Melody A. Vachon ◽  
Jennifer R. McKelvie ◽  
Josh D. Neufeld
Keyword(s):  
Microbial Community ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Bentonite Clay ◽  
Dna Recovery ◽  
Blocking Agents ◽  
Clay Matrix ◽  
Microbial Dna

ABSTRACT Bentonite clay is an integral component of the engineered barrier system of deep geological repositories (DGRs) that are planned for the long-term storage of high-level radioactive waste. Although nucleic acid extraction and analysis can provide powerful qualitative and quantitative data reflecting the presence, abundance, and functional potential of microorganisms within DGR materials, extraction of microbial DNA from bentonite clay is challenging due to the low biomass and adsorption of nucleic acids to the charged clay matrix. In this study, we used quantitative PCR, gel fingerprinting, and high-throughput sequencing of 16S rRNA gene amplicons to assess DNA extraction efficiency from natural MX-80 bentonite and the same material “spiked” with Escherichia coli genomic DNA. Extraction protocols were tested without additives and with casein and phosphate as blocking agents. Although we demonstrate improved DNA recovery by blocking agents at relatively high DNA spiking concentrations, at relatively low spiking concentrations, we detected a high proportion of contaminant nucleic acids from blocking agents that masked sample-specific microbial profile data. Because bacterial genomic DNA associated with casein preparations was insufficiently removed by UV treatment, casein is not recommended as an additive for DNA extractions from low-biomass samples. Instead, we recommend a kit-based extraction protocol for bentonite clay without additional blocking agents, as tested here and validated with multiple MX-80 bentonite samples, ensuring relatively high DNA recoveries with minimal contamination. IMPORTANCE Extraction of microbial DNA from MX-80 bentonite is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Blocking agents improve DNA recovery, but their impact on microbial community profiles from low-biomass samples has not been characterized well. In this study, we evaluated the effect of casein and phosphate as blocking agents for quantitative recovery of nucleic acids from MX-80 bentonite. Our data justify a simplified framework for analyzing microbial community DNA associated with swelling MX-80 bentonite samples within the context of a deep geological repository for used nuclear fuel. This study is among the first to demonstrate successful extraction of DNA from Wyoming MX-80 bentonite.

scholarly journals Different Methods of Soil DNA Extraction

BIO-PROTOCOL ◽  
2020 ◽  
Vol 10 (2) ◽  
Author(s):  
Asmita Kamble ◽  
Harinder Singh
Keyword(s):  
Dna Extraction ◽  
Soil Dna ◽  
Soil Dna Extraction

scholarly journals Innovative Methods for Soil DNA Purification Tested in Soils with Widely Differing Characteristics

2008 ◽  
Vol 74 (9) ◽  
pp. 2902-2907 ◽  
Author(s):  
Marketa Sagova-Mareckova ◽  
Ladislav Cermak ◽  
Jitka Novotna ◽  
Kamila Plhackova ◽  
Jana Forstova ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Soil Ph ◽  
Vegetation Cover ◽  
Organic Matter Content ◽  
Clay Content ◽  
Matter Content ◽  
Soil Dna ◽  
Innovative Methods ◽  
Soil Dna Extraction ◽  
Extraction And Purification

ABSTRACT Seven methods of soil DNA extraction and purification were tested in a set of 14 soils differing in bedrock, texture, pH, salinity, moisture, organic matter content, and vegetation cover. The methods introduced in this study included pretreatment of soil with CaCO3 or purification of extracted DNA by CaCl2. The performance of innovated methods was compared to that of the commercial kit Mo Bio PowerSoil and the phenol-chloroform-based method of D. N. Miller, J. E. Bryant, E. L. Madsen, and W. C. Ghiorse (Appl. Environ. Microbiol. 65:4715-4724, 1999). This study demonstrated significant differences between the tested methods in terms of DNA yield, PCR performance, and recovered bacterial diversity. The differences in DNA yields were correlated to vegetation cover, soil pH, and clay content. The differences in PCR performances were correlated to vegetation cover and soil pH. The innovative methods improved PCR performance in our set of soils, in particular for forest acidic soils. PCR was successful in 95% of cases by the method using CaCl2 purification and in 93% of cases by the method based on CaCO3 pretreatment, but only in 79% by Mo Bio PowerSoil, for our range of soils. Also, the innovative methods recovered a higher percentage of actinomycete diversity from a subset of three soils. Recommendations include the assessment of soil characteristics prior to selecting the optimal protocol for soil DNA extraction and purification.

scholarly journals Sensitive diagnosis of cutaneous leishmaniasis by lesion swab sampling coupled to qPCR

Parasitology ◽  
2014 ◽  
Vol 141 (14) ◽  
pp. 1891-1897 ◽  
Author(s):  
EMILY R. ADAMS ◽  
MARIA ADELAIDA GOMEZ ◽  
LAURA SCHESKE ◽  
RUBY RIOS ◽  
RICARDO MARQUEZ ◽  
...  
Keyword(s):  
Cutaneous Leishmaniasis ◽  
Dna Extraction ◽  
Extraction Methods ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Sample Type ◽  
Recovery Method ◽  
Dna Recovery ◽  
Efficient Recovery ◽  
Dna Extraction Methods

SUMMARYVariation in clinical accuracy of molecular diagnostic methods for cutaneous leishmaniasis (CL) is commonly observed depending on the sample source, the method of DNA recovery and the molecular test. Few attempts have been made to compare these variables. Two swab and aspirate samples from lesions of patients with suspected CL (n = 105) were evaluated alongside standard diagnosis by microscopic detection of amastigotes or culture of parasites from lesion material. Three DNA extraction methods were compared: Qiagen on swab and aspirate specimens, Isohelix on swabs and Boil/Spin of lesion aspirates. Recovery ofLeishmaniaDNA was evaluated for each sample type by real-time polymerase chain reaction detection of parasitic 18S rDNA, and the diagnostic accuracy of the molecular method determined. Swab sampling combined with Qiagen DNA extraction was the most efficient recovery method forLeishmaniaDNA, and was the most sensitive (98%; 95% CI: 91–100%) and specific (84%; 95% CI: 64–95%) approach. Aspirated material was less sensitive at 80% (95% CI: 70–88%) and 61% (95% CI: 50–72%) when coupled to Qiagen or Boil-Spin DNA extraction, respectively. Swab sampling of lesions was painless, simple to perform and coupled with standardized DNA extraction enhances the feasibility of molecular diagnosis of CL.

scholarly journals Toward Best Practice in Livestock Microbiota Research: A Comprehensive Comparison of Sample Storage and DNA Extraction Strategies

2021 ◽  
Vol 12 ◽  
Author(s):  
Gertrude Wegl ◽  
Nikolaus Grabner ◽  
Andreas Köstelbauer ◽  
Viviana Klose ◽  
Mahdi Ghanbari
Keyword(s):  
Microbial Community ◽  
Gut Microbiota ◽  
Dna Extraction ◽  
Best Practice ◽  
Extraction Methods ◽  
Sample Type ◽  
Dependent Manner ◽  
Research Projects ◽  
Sample Storage ◽  
Dna Recovery

Understanding the roles of microorganisms in the animal gastrointestinal microenvironment is highly important for the development of effective strategies to manage and manipulate these microbial communities. In order to guide future animal gut microbiota research projects and standardization efforts, we have conducted a systematic comparison of 10 currently used sample preservation and DNA extraction approaches for pig and chicken microbiota samples and quantified their effects on bacterial DNA yield, quality, integrity, and on the resulting sequence-based bacterial composition estimates. The results showed how key stages of conducting a microbiota study, including the sample storage and DNA extraction, can substantially affect DNA recovery from the microbial community, and therefore, biological interpretation in a matrix-dependent manner. Our results highlight the fact that the influence of storage and extraction methods on the resulting microbial community structure differed by sample type, even within the same species. As the effects of these technical steps are potentially large compared with the real biological variability to be explained, standardization is crucial for accelerating progress in the area of livestock microbiota research. This study provided a framework to assist future animal gut microbiota research projects and standardization efforts.

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