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Sample records for soil dna extracts

  1. Methods for microbial DNA extraction from soil for PCR amplification

    Directory of Open Access Journals (Sweden)

    Yeates C

    1998-01-01

    Full Text Available Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1. DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size.

  2. Necessity of purification during bacterial DNA extraction with environmental soils

    Directory of Open Access Journals (Sweden)

    Hyun Jeong Lim

    2017-08-01

    Full Text Available Complexity and heterogeneity of soil samples have often implied the inclusion of purification steps in conventional DNA extraction for polymerase chain reaction (PCR assays. Unfortunately the purification steps are also time and labor intensive. Therefore the necessity of DNA purification was re-visited and investigated for a variety of environmental soil samples that contained various amounts of PCR inhibitors. Bead beating and centrifugation was used as the baseline (without purification method for DNA extraction. Its performance was compared with that of conventional DNA extraction kit (with purification. The necessity criteria for DNA purification were established with environmental soil samples. Using lysis conditions at 3000 rpm for 3 minutes with 0.1 mm glass beads, centrifugation time of 10 minutes and 1:10 dilution ratio, the baseline method outperformed conventional DNA extraction on cell seeded sand samples. Further investigation with PCR inhibitors (i.e., humic acids, clay, and magnesium [Mg] showed that sand samples containing less than 10 μg/g humic acids and 70% clay may not require purifications. Interestingly, the inhibition pattern of Mg ion was different from other inhibitors due to the complexation interaction of Mg ion with DNA fragments. It was concluded that DNA extraction method without purification is suitable for soil samples that have less than 10 μg/g of humic acids, less than 70% clay content and less than 0.01% Mg ion content.

  3. Comparison of DNA extraction protocols for microbial communities from soil treated with biochar

    Science.gov (United States)

    Leite, D.C.A.; Balieiro, F.C.; Pires, C.A.; Madari, B.E.; Rosado, A.S.; Coutinho, H.L.C.; Peixoto, R.S.

    2014-01-01

    Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit), the PowerSoil® DNA Isolation Kit (PS kit) and the ZR Soil Microbe DNA Kit Miniprep™ (ZR kit), for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits, the PS kit was the most efficient with respect to the amount and purity of recovered DNA and considering the complexity of the generated DGGE microbial fingerprint from the sand-biochar samples. PMID:24948928

  4. Comparison of DNA extraction protocols for microbial communities from soil treated with biochar

    Directory of Open Access Journals (Sweden)

    D.C.A. Leite

    2014-01-01

    Full Text Available Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit, the PowerSoil® DNA Isolation Kit (PS kit and the ZR Soil Microbe DNA Kit MiniprepTM (ZR kit, for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits, the PS kit was the most efficient with respect to the amount and purity of recovered DNA and considering the complexity of the generated DGGE microbial fingerprint from the sand-biochar samples.

  5. The validation of forensic DNA extraction systems to utilize soil contaminated biological evidence.

    Science.gov (United States)

    Kasu, Mohaimin; Shires, Karen

    2015-07-01

    The production of full DNA profiles from biological evidence found in soil has a high failure rate due largely to the inhibitory substance humic acid (HA). Abundant in various natural soils, HA co-extracts with DNA during extraction and inhibits DNA profiling by binding to the molecular components of the genotyping assay. To successfully utilize traces of soil contaminated evidence, such as that found at many murder and rape crime scenes in South Africa, a reliable HA removal extraction system would often be selected based on previous validation studies. However, for many standard forensic DNA extraction systems, peer-reviewed publications detailing the efficacy on soil evidence is either lacking or is incomplete. Consequently, these sample types are often not collected or fail to yield suitable DNA material due to the use of unsuitable methodology. The aim of this study was to validate the common forensic DNA collection and extraction systems used in South Africa, namely DNA IQ, FTA elute and Nucleosave for processing blood and saliva contaminated with HA. A forensic appropriate volume of biological evidence was spiked with HA (0, 0.5, 1.5 and 2.5 mg/ml) and processed through each extraction protocol for the evaluation of HA removal using QPCR and STR-genotyping. The DNA IQ magnetic bead system effectively removed HA from highly contaminated blood and saliva, and generated consistently acceptable STR profiles from both artificially spiked samples and crude soil samples. This system is highly recommended for use on soil-contaminated evidence over the cellulose card-based systems currently being preferentially used for DNA sample collection. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  6. Soil DNA extraction procedure influences protist 18S rRNA gene community profiling outcome

    DEFF Research Database (Denmark)

    Santos, Susana S.; Nunes, Ines Marques; Nielsen, Tue K.

    2017-01-01

    Advances in sequencing technologies allow deeper studies of the soil protist diversity and function. However, little attention has been given to the impact of the chosen soil DNA extraction procedure to the overall results. We examined the effect of three acknowledged DNA recovery methods, two...... manual methods (ISOm-11063, GnS-GII) and one commercial kit (MoBio), on soil protist community structures obtained from different sites with different land uses. Results from 18S rRNA gene amplicon sequencing suggest that DNA extraction method significantly affect the replicate homogeneity, the total...... number of operational taxonomic units (OTUs) recovered and the overall taxonomic structure and diversity of soil protist communities. However, DNA extraction effects did not overwhelm the natural variation among samples, as the community data still strongly grouped by geographical location...

  7. Improving Griffith's protocol for co-extraction of microbial DNA and RNA in adsorptive soils

    DEFF Research Database (Denmark)

    Paulin, Mélanie Marie; Nicolaisen, Mette Haubjerg; Jacobsen, Carsten Suhr

    2013-01-01

    Quantification of microbial gene expression is increasingly being used to study key functions in soil microbial communities, yet major limitations still exist for efficient extraction of nucleic acids, especially RNA for transcript analysis, from this complex matrix. We present an improved......-time PCR on both the RNA (after conversion to cDNA) and the DNA fraction of the extracts. Non-adsorptive soils were characterized by low clay content and/or high phosphate content, whereas adsorptive soils had clay contents above 20% and/or a strong presence of divalent Ca in combination with high p......H. Modifications to the co-extraction protocol improved nucleic acid extraction efficiency from all adsorptive soils and were successfully validated by DGGE analysis of the indigenous community based on 16S rRNA gene and transcripts in soils representing low biomass and/or high clay content. This new approach...

  8. Evaluation of the ISO standard 11063 DNA extraction procedure for assessing soil microbial abundance and community structure.

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    Pierre Plassart

    Full Text Available Soil DNA extraction has become a critical step in describing microbial biodiversity. Historically, ascertaining overarching microbial ecological theories has been hindered as independent studies have used numerous custom and commercial DNA extraction procedures. For that reason, a standardized soil DNA extraction method (ISO-11063 was previously published. However, although this ISO method is suited for molecular tools such as quantitative PCR and community fingerprinting techniques, it has only been optimized for examining soil bacteria. Therefore, the aim of this study was to assess an appropriate soil DNA extraction procedure for examining bacterial, archaeal and fungal diversity in soils of contrasting land-use and physico-chemical properties. Three different procedures were tested: the ISO-11063 standard; a custom procedure (GnS-GII; and a modified ISO procedure (ISOm which includes a different mechanical lysis step (a FastPrep ®-24 lysis step instead of the recommended bead-beating. The efficacy of each method was first assessed by estimating microbial biomass through total DNA quantification. Then, the abundances and community structure of bacteria, archaea and fungi were determined using real-time PCR and terminal restriction fragment length polymorphism approaches. Results showed that DNA yield was improved with the GnS-GII and ISOm procedures, and fungal community patterns were found to be strongly dependent on the extraction method. The main methodological factor responsible for differences between extraction procedure efficiencies was found to be the soil homogenization step. For integrative studies which aim to examine bacteria, archaea and fungi simultaneously, the ISOm procedure results in higher DNA recovery and better represents microbial communities.

  9. Comparative analysis of environmental DNA extraction and purification methods from different humic acid rich soils

    CSIR Research Space (South Africa)

    Lakay, FM

    2007-01-01

    Full Text Available Three different soil DNA isolation and four purification strategies were compared on different soil samples with variable rates of success. Bead beating extraction gave significantly higher DNA yields than microwave-based and liquid nitrogen...

  10. A simple method for normalization of DNA extraction to improve the quantitative detection of soil-borne plant pathogenic oomycetes by real-time PCR.

    Science.gov (United States)

    Li, M; Ishiguro, Y; Kageyama, K; Zhu, Z

    2015-08-01

    Most of the current research into the quantification of soil-borne pathogenic oomycetes lacks determination of DNA extraction efficiency, probably leading to an incorrect estimation of DNA quantity. In this study, we developed a convenient method by using a 100 bp artificially synthesized DNA sequence derived from the mitochondrion NADH dehydrogenase subunit 2 gene of Thunnus thynnus as a control to determine the DNA extraction efficiency. The control DNA was added to soils and then co-extracted along with soil genomic DNA. DNA extraction efficiency was determined by the control DNA. Two different DNA extraction methods were compared and evaluated using different types of soils, and the commercial kit was proved to give more consistent results. We used the control DNA combined with real-time PCR to quantify the oomycete DNAs from 12 naturally infested soils. Detectable target DNA concentrations were three to five times higher after normalization. Our tests also showed that the extraction efficiencies varied on a sample-to-sample basis and were simple and useful for the accurate quantification of soil-borne pathogenic oomycetes. Oomycetes include many important plant pathogens. Accurate quantification of these pathogens is essential in the management of diseases. This study reports an easy method utilizing an external DNA control for the normalization of DNA extraction by real-time PCR. By combining two different efficient soil DNA extraction methods, the developed quantification method dramatically improved the results. This study also proves that the developed normalization method is necessary and useful for the accurate quantification of soil-borne plant pathogenic oomycetes. © 2015 The Society for Applied Microbiology.

  11. DNA analysis of soil extracts can be used to investigate fine root depth distribution of trees

    Science.gov (United States)

    Bithell, Sean L.; Tran-Nguyen, Lucy T. T.; Hearnden, Mark N.; Hartley, Diana M.

    2015-01-01

    Understanding the root distribution of trees by soil coring is time-consuming as it requires the separation of roots from soil and classification of roots into particular size classes. This labour-intensive process can limit sample throughput and therefore sampling intensity. We investigated the use of quantitative polymerase chain reaction (qPCR) on soil DNA extractions to determine live fine root DNA density (RDD, mg DNA m−2) for mango (Mangifera indica) trees. The specificity of the qPCR was tested against DNA extracted from 10 mango cultivars and 14 weed species. All mango cultivars and no weeds were detected. Mango DNA was successfully quantified from control soil spiked with mango roots and weed species. The DNA yield of mango root sections stored in moist soil at 23–28 °C declined after 15 days to low concentrations as roots decayed, indicating that dead root materials in moist soil would not cause false-positive results. To separate large roots from samples, a root separation method for field samples was used to target the root fragments remaining in sieved (minimum 2 mm aperture) soil for RDD comparisons. Using this method we compared the seasonal RDD values of fine roots for five mango rootstock cultivars in a field trial. The mean cultivar DNA yields by depth from root fragments in the sieved soil samples had the strongest relationship (adjusted multiple R2 = 0.9307, P < 0.001) with the dry matter (g m−2) of fine (diameter <0.64 mm) roots removed from the soil by sieving. This method provides a species-specific and rapid means of comparing the distribution and concentration of live fine roots of trees in orchards using soil samples up to 500 g. PMID:25552675

  12. Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil

    Energy Technology Data Exchange (ETDEWEB)

    Hjort, K.; Bergstrom, M.; Adesina, M.F.; Jansson, J.K.; Smalla, K.; Sjoling, S.

    2009-09-01

    Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal-restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal-restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF{sup 103} of the isolate, Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

  13. Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples.

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    Miller, D N; Bryant, J E; Madsen, E L; Ghiorse, W C

    1999-11-01

    We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed

  14. Potential use of DNA adducts to detect mutagenic compounds in soil

    International Nuclear Information System (INIS)

    Hua Guoxiong; Lyons, Brett; Killham, Ken; Singleton, Ian

    2009-01-01

    In this study, three different soils with contrasting features, spiked with 300 mg benzo[a]pyrene (BaP)/kg dry soil, were incubated at 20 deg. C and 60% water holding capacity for 540 days. At different time points, BaP and DNA were extracted and quantified, and DNA adducts were quantified by 32 P-postlabelling. After 540 days incubation, 69.3, 81.6 and 83.2% of initial BaP added remained in Cruden Bay, Boyndie and Insch soils, respectively. Meanwhile, a significantly different amount of DNA-BaP adducts were found in the three soils exposed to BaP over time. The work demonstrates the concept that DNA adducts can be detected on DNA extracted from soil. Results suggest the technique is not able to directly reflect bioavailability of BaP transformation products. However, this new method provides a potential way to detect mutagenic compounds in contaminated soil and to assess the outcomes of soil remediation. - A novel DNA adduct assay may provide a potential technique to detect mutagenic compounds in contaminated soil

  15. Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification ▿

    Science.gov (United States)

    Yankson, Kweku K.; Steck, Todd R.

    2009-01-01

    We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, μg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil. PMID:19633108

  16. A DNA based method to detect the grapevine root-rotting fungus Roesleria subterranea in soil and root samples

    Directory of Open Access Journals (Sweden)

    S. Neuhauser

    2009-05-01

    Full Text Available Roesleria subterranea causes root rot in grapevine and fruit trees. The fungus has long been underestimated as a weak parasite, but during the last years it has been reported to cause severe damages in German vineyards. Direct, observation-based detection of the parasite is time consuming and destructive, as large parts of the rootstocks have to be uprooted and screened for the tiny, stipitate, hypogeous ascomata of R. subterranea. To facilitate rapid detection in vineyards, protocols to extract DNA from soil samples and grapevine roots, and R.-subterranea-specific PCR primers were designed. Twelve DNA-extraction protocols for soil samples were tested in small-scale experiments, and selected parameters were optimised. A protocol based on ball-mill homogenization, DNA extraction with SDS, skim milk, chloroform, and isopropanol, and subsequent purifi cation of the raw extracts with PVPP-spin-columns was most effective. This DNA extraction protocol was found to be suitable for a wide range of soil-types including clay, loam and humic-rich soils. For DNA extraction from grapevine roots a CTAB-based protocol was more reliable for various grapevine rootstock varieties. Roesleria-subterranea-specific primers for the ITS1-5.8S-ITS2 rDNA region were developed and tested for their specifi city to DNA extracts from eleven R. subterranea strains isolated from grapevine and fruit trees. No cross reactions were detected with DNA extracts from 44 different species of fungi isolated from vineyard soils. The sensitivity of the species-specifi c primers in combination with the DNA extraction method for soil was high: as little as 100 fg μl-1 R.-subterranea-DNA was suffi cient for a detection in soil samples and plant material. Given that specifi c primers are available, the presented method will also allow quick and large-scale testing for other root pathogens.

  17. Modification of gDNA extraction from soil for PCR designed for the routine examination of soil samples contaminated with Toxocara spp. eggs.

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    Borecka, A; Gawor, J

    2008-06-01

    A modification of gDNA extraction was developed for the polymerase chain reaction (PCR) technique, intended for the detection and differentiation of Toxocara spp. eggs in soil or sediments. Sand samples from sandpits confirmed as being contaminated with Toxocara spp. eggs by the flotation technique were analysed by PCR. The use of proteinase K made it possible to obtain genomic DNA from the sample without needing to isolate eggs using flotation or to inactivate PCR inhibitors present in the sand. Specific primers in the PCR reaction allowed discrimination between T. canis and T. cati eggs. The modification simplified the procedure, thanks to eliminating the step of gDNA isolation from eggs, which is both laborious and difficult.

  18. Skeleton versus fine earth: what information is stored in the mobile extracellular soil DNA fraction?

    Science.gov (United States)

    Ascher, Judith; Ceccherini, Maria Teresa; Agnelli, Alberto; Corti, Guiseppe; Pietramellara, Giacomo

    2010-05-01

    The soil genome consists of an intracellular and an extracellular fraction. Recently, soil extracellular DNA (eDNA) has been shown to be quantitatively relevant, with a high survival capacity and mobility, playing a crucial role in the gene transfer by transformation, in the formation of bacterial biofilm and as a source of nutrients for soil microorganisms. The eDNA fraction can be discriminated and classified by its interaction with clay minerals, humic acids and Al/Fe oxihydroxides, resulting in differently mobile components. The eDNA extractable in water, classified as DNA free in the extracellular soil environment or adsorbed on soil colloids (eDNAfree/adsorbed), is hypothesized to be the most mobile DNA in soil. Challenging to assess the information stored in this DNA fraction, eDNAfree/adsorbed was recovered from fine earth (gel electrophoresis), and qualitative analysis in terms of the composition and distribution of fungal and bacterial communities (Denaturing Gradient Gel Electrophoresis- fingerprinting). The mobile soil eDNA, extracted from each horizon, was characterised by low molecular weight (result of the movement of eDNA along the soil profile and from fine earth to skeleton. The molecular characterization provided information about the autochthonous microflora inhabiting skeleton and fine earth as well as information about the fate of soil DNA in terms of presence, persistence and movement of eDNA and the stored genetic information.

  19. Nucleic Acid Extraction from Synthetic Mars Analog Soils for in situ Life Detection

    Science.gov (United States)

    Mojarro, Angel; Ruvkun, Gary; Zuber, Maria T.; Carr, Christopher E.

    2017-08-01

    Biological informational polymers such as nucleic acids have the potential to provide unambiguous evidence of life beyond Earth. To this end, we are developing an automated in situ life-detection instrument that integrates nucleic acid extraction and nanopore sequencing: the Search for Extra-Terrestrial Genomes (SETG) instrument. Our goal is to isolate and determine the sequence of nucleic acids from extant or preserved life on Mars, if, for example, there is common ancestry to life on Mars and Earth. As is true of metagenomic analysis of terrestrial environmental samples, the SETG instrument must isolate nucleic acids from crude samples and then determine the DNA sequence of the unknown nucleic acids. Our initial DNA extraction experiments resulted in low to undetectable amounts of DNA due to soil chemistry-dependent soil-DNA interactions, namely adsorption to mineral surfaces, binding to divalent/trivalent cations, destruction by iron redox cycling, and acidic conditions. Subsequently, we developed soil-specific extraction protocols that increase DNA yields through a combination of desalting, utilization of competitive binders, and promotion of anaerobic conditions. Our results suggest that a combination of desalting and utilizing competitive binders may establish a "universal" nucleic acid extraction protocol suitable for analyzing samples from diverse soils on Mars.

  20. Microbial food safety: Potential of DNA extraction methods for use in diagnostic metagenomics

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hasseldam; Andersen, Sandra Christine; Christensen, Julia

    2015-01-01

    ) yielding protocols. The PowerLyzer PowerSoil DNA Isolation Kit performed significantly better than all other protocols tested. Selected protocols were modified, i.e., extended heating and homogenization, resulting in increased yields of total DNA. For QIAamp Fast DNA Stool Mini Kit (Qiagen) a 7-fold...... of the protocols to extract DNA was observed. The highest DNA yield was obtained with the PowerLyzer PowerSoil DNA Isolation Kit, whereas the FastDNA SPIN Kit for Feces (MP Biomedicals) resulted in the highest amount of PCR-amplifiable C. jejuni DNA....

  1. Nucleic Acid Extraction from Synthetic Mars Analog Soils for in situ Life Detection.

    Science.gov (United States)

    Mojarro, Angel; Ruvkun, Gary; Zuber, Maria T; Carr, Christopher E

    2017-08-01

    Biological informational polymers such as nucleic acids have the potential to provide unambiguous evidence of life beyond Earth. To this end, we are developing an automated in situ life-detection instrument that integrates nucleic acid extraction and nanopore sequencing: the Search for Extra-Terrestrial Genomes (SETG) instrument. Our goal is to isolate and determine the sequence of nucleic acids from extant or preserved life on Mars, if, for example, there is common ancestry to life on Mars and Earth. As is true of metagenomic analysis of terrestrial environmental samples, the SETG instrument must isolate nucleic acids from crude samples and then determine the DNA sequence of the unknown nucleic acids. Our initial DNA extraction experiments resulted in low to undetectable amounts of DNA due to soil chemistry-dependent soil-DNA interactions, namely adsorption to mineral surfaces, binding to divalent/trivalent cations, destruction by iron redox cycling, and acidic conditions. Subsequently, we developed soil-specific extraction protocols that increase DNA yields through a combination of desalting, utilization of competitive binders, and promotion of anaerobic conditions. Our results suggest that a combination of desalting and utilizing competitive binders may establish a "universal" nucleic acid extraction protocol suitable for analyzing samples from diverse soils on Mars. Key Words: Life-detection instruments-Nucleic acids-Mars-Panspermia. Astrobiology 17, 747-760.

  2. Meta-barcoding of 'dirt' DNA from soil reflects vertebrate biodiversity.

    Science.gov (United States)

    Andersen, Kenneth; Bird, Karen Lise; Rasmussen, Morten; Haile, James; Breuning-Madsen, Henrik; Kjaer, Kurt H; Orlando, Ludovic; Gilbert, M Thomas P; Willerslev, Eske

    2012-04-01

    DNA molecules originating from animals and plants can be retrieved directly from sediments and have been used for reconstructing both contemporary and past ecosystems. However, the extent to which such 'dirt' DNA reflects taxonomic richness and structural diversity remains contentious. Here, we couple second generation high-throughput sequencing with 16S mitochondrial DNA (mtDNA) meta-barcoding, to explore the accuracy and sensitivity of 'dirt' DNA as an indicator of vertebrate diversity, from soil sampled at safari parks, zoological gardens and farms with known species compositions. PCR amplification was successful in the full pH range of the investigated soils (6.2 ± 0.2 to 8.3 ± 0.2), but inhibition was detected in extracts from soil of high organic content. DNA movement (leaching) through strata was evident in some sporadic cases and is influenced by soil texture and structure. We find that DNA from the soil surface reflects overall taxonomic richness and relative biomass of individual species. However, one species that was recently introduced was not detected. Furthermore, animal behaviour was shown to influence DNA deposition rates. The approach potentially provides a quick methodological alternative to classical ecological surveys of biodiversity, and most reliable results are obtained with spatial sample replicates, while relative amounts of soil processed per site is of less importance. © 2011 Blackwell Publishing Ltd.

  3. Efficient recovery of environmental DNA for expression cloning by indirect extraction methods

    NARCIS (Netherlands)

    Gabor, Esther; de Vries, Erik; Janssen, DB

    2003-01-01

    Using direct and cell extraction-based (indirect) isolation methods, DNA was obtained from environmental samples with largely differing characteristics (loam soil, sand soil, sediment, activated sludge, and compost) and evaluated with respect to the comprised bacterial diversity and its suitability

  4. New environmental metabarcodes for analysing soil DNA

    DEFF Research Database (Denmark)

    Epp, Laura S.; Boessenkool, Sanne; Bellemain, Eva P.

    2012-01-01

    was systematically evaluated by (i) in silico PCRs using all standard sequences in the EMBL public database as templates, (ii) in vitro PCRs of DNA extracts from surface soil samples from a site in Varanger, northern Norway and (iii) in vitro PCRs of DNA extracts from permanently frozen sediment samples of late......Metabarcoding approaches use total and typically degraded DNA from environmental samples to analyse biotic assemblages and can potentially be carried out for any kinds of organisms in an ecosystem. These analyses rely on specific markers, here called metabarcodes, which should be optimized...... for taxonomic resolution, minimal bias in amplification of the target organism group and short sequence length. Using bioinformatic tools, we developed metabarcodes for several groups of organisms: fungi, bryophytes, enchytraeids, beetles and birds. The ability of these metabarcodes to amplify the target groups...

  5. Direct quantification of fungal DNA from soil substrate using real-time PCR.

    Science.gov (United States)

    Filion, Martin; St-Arnaud, Marc; Jabaji-Hare, Suha H

    2003-04-01

    Detection and quantification of genomic DNA from two ecologically different fungi, the plant pathogen Fusarium solani f. sp. phaseoli and the arbuscular mycorrhizal fungus Glomus intraradices, was achieved from soil substrate. Specific primers targeting a 362-bp fragment from the SSU rRNA gene region of G. intraradices and a 562-bp fragment from the F. solani f. sp. phaseoli translation elongation factor 1 alpha gene were used in real-time polymerase chain reaction (PCR) assays conjugated with the fluorescent SYBR(R) Green I dye. Standard curves showed a linear relation (r(2)=0.999) between log values of fungal genomic DNA of each species and real-time PCR threshold cycles and were quantitative over 4-5 orders of magnitude. Real-time PCR assays were applied to in vitro-produced fungal structures and sterile and non-sterile soil substrate seeded with known propagule numbers of either fungi. Detection and genomic DNA quantification was obtained from the different treatments, while no amplicon was detected from non-seeded non-sterile soil samples, confirming the absence of cross-reactivity with the soil microflora DNA. A significant correlation (Pgenomic DNA of F. solani f. sp. phaseoli or G. intraradices detected and the number of fungal propagules present in seeded soil substrate. The DNA extraction protocol and real-time PCR quantification assay can be performed in less than 2 h and is adaptable to detect and quantify genomic DNA from other soilborne fungi.

  6. Repeated extraction of DNA from FTA cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Ferrero, Laura; Børsting, Claus

    2011-01-01

    Extraction of DNA using magnetic bead based techniques on automated DNA extraction instruments provides a fast, reliable and reproducible method for DNA extraction from various matrices. However, the yield of extracted DNA from FTA-cards is typically low. Here, we demonstrate that it is possible...... to repeatedly extract DNA from the processed FTA-disk. The method increases the yield from the nanogram range to the microgram range....

  7. Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes.

    Science.gov (United States)

    Yamagishi, Junya; Sato, Yukuto; Shinozaki, Natsuko; Ye, Bin; Tsuboi, Akito; Nagasaki, Masao; Yamashita, Riu

    2016-01-01

    The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no "gold standard" for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study.

  8. Evaluation of DNA extraction methods for PCR-based detection of Listeria monocytogenes from vegetables.

    Science.gov (United States)

    Vojkovska, H; Kubikova, I; Kralik, P

    2015-03-01

    Epidemiological data indicate that raw vegetables are associated with outbreaks of Listeria monocytogenes. Therefore, there is a demand for the availability of rapid and sensitive methods, such as PCR assays, for the detection and accurate discrimination of L. monocytogenes. However, the efficiency of PCR methods can be negatively affected by inhibitory compounds commonly found in vegetable matrices that may cause false-negative results. Therefore, the sample processing and DNA isolation steps must be carefully evaluated prior to the introduction of such methods into routine practice. In this study, we compared the ability of three column-based and four magnetic bead-based commercial DNA isolation kits to extract DNA of the model micro-organism L. monocytogenes from raw vegetables. The DNA isolation efficiency of all isolation kits was determined using a triplex real-time qPCR assay designed to specifically detect L. monocytogenes. The kit with best performance, the PowerSoil(™) Microbial DNA Isolation Kit, is suitable for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. Coupled with the triplex real-time qPCR assay, this DNA isolation kit is applicable to the samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. Several recent outbreaks of Listeria monocytogenes have been associated with the consumption of fruits and vegetables. Real-time PCR assays allow fast detection and accurate quantification of microbes. However, the success of real-time PCR is dependent on the success with which template DNA can be extracted. The results of this study suggest that the PowerSoil(™) Microbial DNA Isolation Kit can be used for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. This method is applicable to samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. © 2014

  9. Validation of a DNA IQ-based extraction method for TECAN robotic liquid handling workstations for processing casework.

    Science.gov (United States)

    Frégeau, Chantal J; Lett, C Marc; Fourney, Ron M

    2010-10-01

    A semi-automated DNA extraction process for casework samples based on the Promega DNA IQ™ system was optimized and validated on TECAN Genesis 150/8 and Freedom EVO robotic liquid handling stations configured with fixed tips and a TECAN TE-Shake™ unit. The use of an orbital shaker during the extraction process promoted efficiency with respect to DNA capture, magnetic bead/DNA complex washes and DNA elution. Validation studies determined the reliability and limitations of this shaker-based process. Reproducibility with regards to DNA yields for the tested robotic workstations proved to be excellent and not significantly different than that offered by the manual phenol/chloroform extraction. DNA extraction of animal:human blood mixtures contaminated with soil demonstrated that a human profile was detectable even in the presence of abundant animal blood. For exhibits containing small amounts of biological material, concordance studies confirmed that DNA yields for this shaker-based extraction process are equivalent or greater to those observed with phenol/chloroform extraction as well as our original validated automated magnetic bead percolation-based extraction process. Our data further supports the increasing use of robotics for the processing of casework samples. Crown Copyright © 2009. Published by Elsevier Ireland Ltd. All rights reserved.

  10. Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes.

    Directory of Open Access Journals (Sweden)

    Junya Yamagishi

    Full Text Available The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field, QIAsymphony (a robotics method, and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no "gold standard" for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study.

  11. Automated extraction of DNA from clothing

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Hjort, Benjamin Benn; Nøhr Hansen, Thomas

    2011-01-01

    Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing...

  12. High-throughput sequencing of nematode communities from total soil DNA extractions

    DEFF Research Database (Denmark)

    Sapkota, Rumakanta; Nicolaisen, Mogens

    2015-01-01

    nematodes without the need for enrichment was developed. Using this strategy on DNA templates from a set of 22 agricultural soils, we obtained 64.4% sequences of nematode origin in total, whereas the remaining sequences were almost entirely from other metazoans. The nematode sequences were derived from...... in previous sequence-based studies are not nematode specific but also amplify other groups of organisms such as fungi and plantae, and thus require a nematode enrichment step that may introduce biases. Results: In this study an amplification strategy which selectively amplifies a fragment of the SSU from...... a broad taxonomic range and most sequences were from nematode taxa that have previously been found to be abundant in soil such as Tylenchida, Rhabditida, Dorylaimida, Triplonchida and Araeolaimida. Conclusions: Our amplification and sequencing strategy for assessing nematode diversity was able to collect...

  13. DNA damage and repair process in earthworm after in-vivo and in vitro exposure to soils irrigated by wastewaters

    International Nuclear Information System (INIS)

    Qiao Min; Chen Ying; Wang Chunxia; Wang Zijian; Zhu Yongguan

    2007-01-01

    In this study, DNA damage to earthworms (Eisenia fetida) after in vivo exposure to contaminated soils was measured by detecting DNA strand breakages (DSBs) and causality was analyzed through fractionation based bioassays. A non-linear dose-response relationship existed between DNA damage and total soil PAHs levels. DNA damage, measured with the comet assay, and its repair process, were observed. To identify the chemical causality, an in vitro comet assay using coelomocytes was subsequently performed on the fractionated organic extracts from soils. The results showed that the PAHs in the soils were responsible for the exerting genotoxic effects on earthworms. When normalized to benzo(a)pyrene toxic equivalent (TEQ BaP ), the saturation dose in the dose-response curve was about 10 ng TEQ BaP g -1 soil (dw). - A non-linear dose-response relationship exists between earthworm DNA damage, measured with comet assay, and total PAHs levels in soils irrigated by wastewaters

  14. DNA damage and repair process in earthworm after in-vivo and in vitro exposure to soils irrigated by wastewaters

    Energy Technology Data Exchange (ETDEWEB)

    Qiao Min [State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Chen Ying [State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Wang Chunxia [State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Wang Zijian [State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China)]. E-mail: wangzj@rcees.ac.cn; Zhu Yongguan [State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China)

    2007-07-15

    In this study, DNA damage to earthworms (Eisenia fetida) after in vivo exposure to contaminated soils was measured by detecting DNA strand breakages (DSBs) and causality was analyzed through fractionation based bioassays. A non-linear dose-response relationship existed between DNA damage and total soil PAHs levels. DNA damage, measured with the comet assay, and its repair process, were observed. To identify the chemical causality, an in vitro comet assay using coelomocytes was subsequently performed on the fractionated organic extracts from soils. The results showed that the PAHs in the soils were responsible for the exerting genotoxic effects on earthworms. When normalized to benzo(a)pyrene toxic equivalent (TEQ{sub BaP}), the saturation dose in the dose-response curve was about 10 ng TEQ{sub BaP} g{sup -1} soil (dw). - A non-linear dose-response relationship exists between earthworm DNA damage, measured with comet assay, and total PAHs levels in soils irrigated by wastewaters.

  15. Extraction of ultrashort DNA molecules from herbarium specimens.

    Science.gov (United States)

    Gutaker, Rafal M; Reiter, Ella; Furtwängler, Anja; Schuenemann, Verena J; Burbano, Hernán A

    2017-02-01

    DNA extracted from herbarium specimens is highly fragmented; therefore, it is crucial to use extraction protocols that retrieve short DNA molecules. Improvements in extraction and DNA library preparation protocols for animal remains have allowed efficient retrieval of molecules shorter than 50 bp. Here, we applied these improvements to DNA extraction protocols for herbarium specimens and evaluated extraction performance by shotgun sequencing, which allows an accurate estimation of the distribution of DNA fragment lengths. Extraction with N-phenacylthiazolium bromide (PTB) buffer decreased median fragment length by 35% when compared with cetyl-trimethyl ammonium bromide (CTAB); modifying the binding conditions of DNA to silica allowed for an additional decrease of 10%. We did not observe a further decrease in length for single-stranded DNA (ssDNA) versus double-stranded DNA (dsDNA) library preparation methods. Our protocol enables the retrieval of ultrashort molecules from herbarium specimens, which will help to unlock the genetic information stored in herbaria.

  16. Multilevel soil-vapor extraction test for heterogeneous soil

    International Nuclear Information System (INIS)

    Widdowson, M.A.; Haney, O.R.; Reeves, H.W.

    1997-01-01

    The design, performance, and analysis of a field method for quantifying contaminant mass-extraction rates and air-phase permeability at discrete vertical locations of the vadose zones are presented. The test configuration consists of a multiscreen extraction well and multilevel observation probes located in soil layers adjacent to the extraction well. For each level tested an inflatable packer system is used to pneumatically isolate a single screen in the extraction well, and a vacuum is applied to induce air flow through the screen. Test data include contaminant concentration and flow characteristics at the extraction well, and transient or steady-state pressure drawdown data at observation probes located at variable radii from the extraction well. The test method is applicable to the design of soil-vapor extraction (SVE) and bioventing remediation systems in a variety of geologic settings, particularly stratified soils. Application of the test method at a gasoline-polluted site located in the Piedmont physiographic region is described. Contaminant mass-extraction rates, expressed in terms of volatile hydrocarbons, varied from 0.16 to 14 kg/d

  17. Comparison of Methods of Extracting Salmonella enterica Serovar Enteritidis DNA from Environmental Substrates and Quantification of Organisms by Using a General Internal Procedural Control

    NARCIS (Netherlands)

    Klerks, M.M.; Bruggen, van A.H.C.; Zijlstra, C.; Donnikov, M.; Vos, de R.

    2006-01-01

    This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general

  18. DNA Extraction Techniques for Use in Education

    Science.gov (United States)

    Hearn, R. P.; Arblaster, K. E.

    2010-01-01

    DNA extraction provides a hands-on introduction to DNA and enables students to gain real life experience and practical knowledge of DNA. Students gain a sense of ownership and are more enthusiastic when they use their own DNA. A cost effective, simple protocol for DNA extraction and visualization was devised. Buccal mucosal epithelia provide a…

  19. Some plant extracts retarde nitrification in soil

    Directory of Open Access Journals (Sweden)

    Abdul–Mehdi S. AL-ANSARI

    2015-12-01

    Full Text Available An incubation experiment was conducted to evaluate the effect of aqueous extracts of 17 plant materials on nitrification inhibition of urea- N in soil as compared with chemical inhibitor Dicyandiamide (DCD. Plant materials used in study were collected from different areas of Basrah province, south of Iraq. Aqueous extracts were prepared at ratio of 1:10 (plant material: water and added at conc. of 0.05, 0.10 and 0.20 ml g– 1 soil to loamy sand soil. DCD was added to soil at rate of 50 µg g-1 soil . Soil received urea at rate of 1000 µg N g-1 soil. Treated soils were incubated at 30 OC for 40 days. Results showed that application of all plant extracts, except those of casuarina, date palm and eucalyptus to soil retarded nitrification in soil. Caper, Sowthistle ,bladygrass and pomegranate extracts showed highest inhibition percentage (51, 42, 40 and 40 %, respectively and were found to be more effective than DCD (33 %. Highest inhibition was achieved by using those extracts at conc. of 0.1 ml g-1 soil after 10 days of incubation . Data also revealed that treated soil with these plant extracts significantly increased amount of NH4+–N and decreased amount of NO3-–N accumulation in soil compared with DCD and control treatments. Results of the study suggested a possibility of using aqueous extracts of some studied plants as potent nitrification inhibitor in soil.

  20. Extraction Techniques for Polycyclic Aromatic Hydrocarbons in Soils

    Science.gov (United States)

    Lau, E. V.; Gan, S.; Ng, H. K.

    2010-01-01

    This paper aims to provide a review of the analytical extraction techniques for polycyclic aromatic hydrocarbons (PAHs) in soils. The extraction technologies described here include Soxhlet extraction, ultrasonic and mechanical agitation, accelerated solvent extraction, supercritical and subcritical fluid extraction, microwave-assisted extraction, solid phase extraction and microextraction, thermal desorption and flash pyrolysis, as well as fluidised-bed extraction. The influencing factors in the extraction of PAHs from soil such as temperature, type of solvent, soil moisture, and other soil characteristics are also discussed. The paper concludes with a review of the models used to describe the kinetics of PAH desorption from soils during solvent extraction. PMID:20396670

  1. Extraction Techniques for Polycyclic Aromatic Hydrocarbons in Soils

    Directory of Open Access Journals (Sweden)

    E. V. Lau

    2010-01-01

    Full Text Available This paper aims to provide a review of the analytical extraction techniques for polycyclic aromatic hydrocarbons (PAHs in soils. The extraction technologies described here include Soxhlet extraction, ultrasonic and mechanical agitation, accelerated solvent extraction, supercritical and subcritical fluid extraction, microwave-assisted extraction, solid phase extraction and microextraction, thermal desorption and flash pyrolysis, as well as fluidised-bed extraction. The influencing factors in the extraction of PAHs from soil such as temperature, type of solvent, soil moisture, and other soil characteristics are also discussed. The paper concludes with a review of the models used to describe the kinetics of PAH desorption from soils during solvent extraction.

  2. Extraction of inhibitor-free metagenomic DNA from polluted sediments, compatible with molecular diversity analysis using adsorption and ion-exchange treatments.

    Science.gov (United States)

    Desai, Chirayu; Madamwar, Datta

    2007-03-01

    PCR inhibitor-free metagenomic DNA of high quality and high yield was extracted from highly polluted sediments using a simple remediation strategy of adsorption and ion-exchange chromatography. Extraction procedure was optimized with series of steps, which involved gentle mechanical lysis, treatment with powdered activated charcoal (PAC) and ion-exchange chromatography with amberlite resin. Quality of the extracted DNA for molecular diversity analysis was tested by amplifying bacterial 16S rDNA (16S rRNA gene) with eubacterial specific universal primers (8f and 1492r), cloning of the amplified 16S rDNA and ARDRA (amplified rDNA restriction analysis) of the 16S rDNA clones. The presence of discrete differences in ARDRA banding profiles provided evidence for expediency of the DNA extraction protocol in molecular diversity studies. A comparison of the optimized protocol with commercial Ultraclean Soil DNA isolation kit suggested that method described in this report would be more efficient in removing metallic and organic inhibitors, from polluted sediment samples.

  3. Aluminium, extractable from soil samples by the acid ammonium acetate soil-testing method

    Directory of Open Access Journals (Sweden)

    Osmo Mäkitie

    1968-05-01

    Full Text Available The extractant, 0.5 M acetic acid –0.5 M ammonium acetate at pH 4.65, which is used in soil-testing, extracts relatively high amounts of aluminium from acid soils. The mean values of acetate-extractable aluminium at pH 4.65, 1.75 meq Al/100 g of soil, and of exchangeable aluminium (M KCI extraction, 0.41 meq Al were obtained from a material of 30 samples of acid soils (Table 2. Several other acetic acid ammonium acetate extractants, from M acetic acid to M ammonium acetate solution were also used for studying the extractability of soil aluminium. The soil-testing extractant can be used for the estimation of the soluble amounts of aluminium in acid soils, however, further studies are needed for a better interpretation of the ammonium acetate extractable (at pH 4.65 aluminium in our soils.

  4. Inhibitory and toxic effects of extracellular self-DNA in litter: a mechanism for negative plant-soil feedbacks?

    Science.gov (United States)

    Mazzoleni, Stefano; Bonanomi, Giuliano; Incerti, Guido; Chiusano, Maria Luisa; Termolino, Pasquale; Mingo, Antonio; Senatore, Mauro; Giannino, Francesco; Cartenì, Fabrizio; Rietkerk, Max; Lanzotti, Virginia

    2015-02-01

    Plant-soil negative feedback (NF) is recognized as an important factor affecting plant communities. The objectives of this work were to assess the effects of litter phytotoxicity and autotoxicity on root proliferation, and to test the hypothesis that DNA is a driver of litter autotoxicity and plant-soil NF. The inhibitory effect of decomposed litter was studied in different bioassays. Litter biochemical changes were evaluated with nuclear magnetic resonance (NMR) spectroscopy. DNA accumulation in litter and soil was measured and DNA toxicity was assessed in laboratory experiments. Undecomposed litter caused nonspecific inhibition of root growth, while autotoxicity was produced by aged litter. The addition of activated carbon (AC) removed phytotoxicity, but was ineffective against autotoxicity. Phytotoxicity was related to known labile allelopathic compounds. Restricted (13) C NMR signals related to nucleic acids were the only ones negatively correlated with root growth on conspecific substrates. DNA accumulation was observed in both litter decomposition and soil history experiments. Extracted total DNA showed evident species-specific toxicity. Results indicate a general occurrence of litter autotoxicity related to the exposure to fragmented self-DNA. The evidence also suggests the involvement of accumulated extracellular DNA in plant-soil NF. Further studies are needed to further investigate this unexpected function of extracellular DNA at the ecosystem level and related cellular and molecular mechanisms. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  5. To beat or not to beat a tick: comparison of DNA extraction methods for ticks (Ixodes scapularis

    Directory of Open Access Journals (Sweden)

    Alyssa D. Ammazzalorso

    2015-08-01

    Full Text Available Background. Blacklegged ticks (Ixodes scapularis are important disease vectors in the United States, known to transmit a variety of pathogens to humans, including bacteria, protozoa, and viruses. Their importance as a disease vector necessitates reliable and comparable methods for extracting microbial DNA from ticks. Furthermore, to explore the population genetics or genomics of this tick, appropriate DNA extraction techniques are needed for both the vector and its microbes. Although a few studies have investigated different methods of DNA isolation from ticks, they are limited in the number and types of DNA extraction and lack species-specific quantification of DNA yield.Methods. Here we determined the most efficient and consistent method of DNA extraction from two different developmental stages of I. scapularis—nymph and adult—that are the most important for disease transmission. We used various methods of physical disruption of the hard, chitinous exoskeleton, as well as commercial and non-commercial DNA isolation kits. To gauge the effectiveness of these methods, we quantified the DNA yield and confirmed the DNA quality via PCR of both tick and microbial genetic material.Results. DNA extraction using the Thermo GeneJET Genomic DNA Purification Kit resulted in the highest DNA yields and the most consistent PCR amplification when combined with either cutting or bead beating with select matrices across life stages. DNA isolation methods using ammonium hydroxide as well as the MoBio PowerSoil kit also produced strong and successful PCR amplification, but only for females.Discussion. We contrasted a variety of readily available methods of DNA extraction from single individual blacklegged ticks and presented the results through a quantitative and qualitative assessment.

  6. DNA extraction from herbarium specimens.

    Science.gov (United States)

    Drábková, Lenka Záveská

    2014-01-01

    With the expansion of molecular techniques, the historical collections have become widely used. Studying plant DNA using modern molecular techniques such as DNA sequencing plays an important role in understanding evolutionary relationships, identification through DNA barcoding, conservation status, and many other aspects of plant biology. Enormous herbarium collections are an important source of material especially for specimens from areas difficult to access or from taxa that are now extinct. The ability to utilize these specimens greatly enhances the research. However, the process of extracting DNA from herbarium specimens is often fraught with difficulty related to such variables as plant chemistry, drying method of the specimen, and chemical treatment of the specimen. Although many methods have been developed for extraction of DNA from herbarium specimens, the most frequently used are modified CTAB and DNeasy Plant Mini Kit protocols. Nine selected protocols in this chapter have been successfully used for high-quality DNA extraction from different kinds of plant herbarium tissues. These methods differ primarily with respect to their requirements for input material (from algae to vascular plants), type of the plant tissue (leaves with incrustations, sclerenchyma strands, mucilaginous tissues, needles, seeds), and further possible applications (PCR-based methods or microsatellites, AFLP).

  7. The persistence of human DNA in soil following surface decomposition.

    Science.gov (United States)

    Emmons, Alexandra L; DeBruyn, Jennifer M; Mundorff, Amy Z; Cobaugh, Kelly L; Cabana, Graciela S

    2017-09-01

    Though recent decades have seen a marked increase in research concerning the impact of human decomposition on the grave soil environment, the fate of human DNA in grave soil has been relatively understudied. With the purpose of supplementing the growing body of literature in forensic soil taphonomy, this study assessed the relative persistence of human DNA in soil over the course of decomposition. Endpoint PCR was used to assess the presence or absence of human nuclear and mitochondrial DNA, while qPCR was used to evaluate the quantity of human DNA recovered from the soil beneath four cadavers at the University of Tennessee's Anthropology Research Facility (ARF). Human nuclear DNA from the soil was largely unrecoverable, while human mitochondrial DNA was detectable in the soil throughout all decomposition stages. Mitochondrial DNA copy abundances were not significantly different between decomposition stages and were not significantly correlated to soil edaphic parameters tested. There was, however, a significant positive correlation between mitochondrial DNA copy abundances and the human associated bacteria, Bacteroides, as estimated by 16S rRNA gene abundances. These results show that human mitochondrial DNA can persist in grave soil and be consistently detected throughout decomposition. Copyright © 2017 The Chartered Society of Forensic Sciences. Published by Elsevier B.V. All rights reserved.

  8. PROTOCOL FOR EXTRACTION OF BACTERIAL METAGENOME DNA TO PRAWN Macrobrachium carcinus L

    Directory of Open Access Journals (Sweden)

    J U González de la Cruz

    2011-07-01

    Full Text Available In this work we adapted a protocol for the extraction of metagenomic DNA (ADNmg bacteria in the digestive system (intestines, stomach and hepatopancreas of Macrobrachium carcinus L., with reference to the method of extracting bacterial DNA from soils and sediments (Rojas-Herrera et al., 2008. This methodology consisted of enzymatic, physics, mechanics and chemistry after a series of tests was abolished enzymatic lysis. However, the success ADNmg extraction was influenced mainly by the preparation of the samples, in particular the hepatopancreas, where it was necessary to remove the fat by thermal shock temperature and phase separation by centrifugation with the sample frozen.The effectiveness of isolated DNA fragmentation was verified by gel electrophoresis in denaturing gradient (DGGE after amplification with universal primers. In general, it had a low diversity (19 phylotypes between the different organs analyzed of 13.5 ± 1 (intestines to 11.7 ± 0.96 (stomach. The Shannon-Weaver index (2.45, Simpsons (10.88 and equity (0972 obtained from the digitization of the image of the gel, suggested that the phylotypes that form the gut microflora M. carcinus, is distributed unevenly between the different organs analyzed.

  9. Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences.

    Science.gov (United States)

    Wagner Mackenzie, Brett; Waite, David W; Taylor, Michael W

    2015-01-01

    The human gut contains dense and diverse microbial communities which have profound influences on human health. Gaining meaningful insights into these communities requires provision of high quality microbial nucleic acids from human fecal samples, as well as an understanding of the sources of variation and their impacts on the experimental model. We present here a systematic analysis of commonly used microbial DNA extraction methods, and identify significant sources of variation. Five extraction methods (Human Microbiome Project protocol, MoBio PowerSoil DNA Isolation Kit, QIAamp DNA Stool Mini Kit, ZR Fecal DNA MiniPrep, phenol:chloroform-based DNA isolation) were evaluated based on the following criteria: DNA yield, quality and integrity, and microbial community structure based on Illumina amplicon sequencing of the V4 region of bacterial and archaeal 16S rRNA genes. Our results indicate that the largest portion of variation within the model was attributed to differences between subjects (biological variation), with a smaller proportion of variation associated with DNA extraction method (technical variation) and intra-subject variation. A comprehensive understanding of the potential impact of technical variation on the human gut microbiota will help limit preventable bias, enabling more accurate diversity estimates.

  10. Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences

    Directory of Open Access Journals (Sweden)

    Brett eWagner Mackenzie

    2015-02-01

    Full Text Available The human gut contains dense and diverse microbial communities which have profound influences on human health. Gaining meaningful insights into these communities requires provision of high quality microbial nucleic acids from human fecal samples, as well as an understanding of the sources of variation and their impacts on the experimental model. We present here a systematic analysis of commonly used microbial DNA extraction methods, and identify significant sources of variation. Five extraction methods (Human Microbiome Project protocol, MoBio PowerSoil DNA Isolation Kit, QIAamp DNA Stool Mini Kit, ZR Fecal DNA MiniPrep, phenol:chloroform-based DNA isolation were evaluated based on the following criteria: DNA yield, quality and integrity, and microbial community structure based on Illumina amplicon sequencing of the V4 region of bacterial and archaeal 16S rRNA genes. Our results indicate that the largest portion of variation within the model was attributed to differences between subjects (biological variation, with a smaller proportion of variation associated with DNA extraction method (technical variation and intra-subject variation. A comprehensive understanding of the potential impact of technical variation on the human gut microbiota will help limit preventable bias, enabling more accurate diversity estimates.

  11. DNA extraction on bio-chip: history and preeminence over conventional and solid-phase extraction methods.

    Science.gov (United States)

    Ayoib, Adilah; Hashim, Uda; Gopinath, Subash C B; Md Arshad, M K

    2017-11-01

    This review covers a developmental progression on early to modern taxonomy at cellular level following the advent of electron microscopy and the advancement in deoxyribonucleic acid (DNA) extraction for expatiation of biological classification at DNA level. Here, we discuss the fundamental values of conventional chemical methods of DNA extraction using liquid/liquid extraction (LLE) followed by development of solid-phase extraction (SPE) methods, as well as recent advances in microfluidics device-based system for DNA extraction on-chip. We also discuss the importance of DNA extraction as well as the advantages over conventional chemical methods, and how Lab-on-a-Chip (LOC) system plays a crucial role for the future achievements.

  12. Comparative analysis of protocols for DNA extraction from soybean caterpillars.

    Science.gov (United States)

    Palma, J; Valmorbida, I; da Costa, I F D; Guedes, J V C

    2016-04-07

    Genomic DNA extraction is crucial for molecular research, including diagnostic and genome characterization of different organisms. The aim of this study was to comparatively analyze protocols of DNA extraction based on cell lysis by sarcosyl, cetyltrimethylammonium bromide, and sodium dodecyl sulfate, and to determine the most efficient method applicable to soybean caterpillars. DNA was extracted from specimens of Chrysodeixis includens and Spodoptera eridania using the aforementioned three methods. DNA quantification was performed using spectrophotometry and high molecular weight DNA ladders. The purity of the extracted DNA was determined by calculating the A260/A280 ratio. Cost and time for each DNA extraction method were estimated and analyzed statistically. The amount of DNA extracted by these three methods was sufficient for PCR amplification. The sarcosyl method yielded DNA of higher purity, because it generated a clearer pellet without viscosity, and yielded high quality amplification products of the COI gene I. The sarcosyl method showed lower cost per extraction and did not differ from the other methods with respect to preparation times. Cell lysis by sarcosyl represents the best method for DNA extraction in terms of yield, quality, and cost effectiveness.

  13. Chelant extraction of heavy metals from contaminated soils

    Energy Technology Data Exchange (ETDEWEB)

    Peters, R.W. [Energy Systems Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439 (United States)

    1999-04-23

    The current state of the art regarding the use of chelating agents to extract heavy metal contaminants has been addressed. Results are presented for treatability studies conducted as worst-case and representative soils from Aberdeen Proving Ground's J-Field for extraction of copper (Cu), lead (Pb), and zinc (Zn). The particle size distribution characteristics of the soils determined from hydrometer tests are approximately 60% sand, 30% silt, and 10% clay. Sequential extractions were performed on the 'as-received' soils (worst case and representative) to determine the speciation of the metal forms. The technique speciates the heavy metal distribution into an easily extractable (exchangeable) form, carbonates, reducible oxides, organically-bound, and residual forms. The results indicated that most of the metals are in forms that are amenable to soil washing (i.e. exchangeable+carbonate+reducible oxides). The metals Cu, Pb, Zn, and Cr have greater than 70% of their distribution in forms amenable to soil washing techniques, while Cd, Mn, and Fe are somewhat less amenable to soil washing using chelant extraction. However, the concentrations of Cd and Mn are low in the contaminated soil. From the batch chelant extraction studies, ethylenediaminetetraacetic acid (EDTA), citric acid, and nitrilotriacetic acid (NTA) were all effective in removing copper, lead, and zinc from the J-Field soils. Due to NTA being a Class II carcinogen, it is not recommended for use in remediating contaminated soils. EDTA and citric acid appear to offer the greatest potential as chelating agents to use in soil washing the Aberdeen Proving Ground soils. The other chelating agents studied (gluconate, oxalate, Citranox, ammonium acetate, and phosphoric acid, along with pH-adjusted water) were generally ineffective in mobilizing the heavy metals from the soils. The chelant solution removes the heavy metals (Cd, Cu, Pb, Zn, Fe, Cr, As, and Hg) simultaneously. Using a multiple

  14. Chelant extraction of heavy metals from contaminated soils

    International Nuclear Information System (INIS)

    Peters, R.W.

    1999-01-01

    The current state of the art regarding the use of chelating agents to extract heavy metal contaminants has been addressed. Results are presented for treatability studies conducted as worst-case and representative soils from Aberdeen Proving Ground's J-Field for extraction of copper (Cu), lead (Pb), and zinc (Zn). The particle size distribution characteristics of the soils determined from hydrometer tests are approximately 60% sand, 30% silt, and 10% clay. Sequential extractions were performed on the 'as-received' soils (worst case and representative) to determine the speciation of the metal forms. The technique speciates the heavy metal distribution into an easily extractable (exchangeable) form, carbonates, reducible oxides, organically-bound, and residual forms. The results indicated that most of the metals are in forms that are amenable to soil washing (i.e. exchangeable+carbonate+reducible oxides). The metals Cu, Pb, Zn, and Cr have greater than 70% of their distribution in forms amenable to soil washing techniques, while Cd, Mn, and Fe are somewhat less amenable to soil washing using chelant extraction. However, the concentrations of Cd and Mn are low in the contaminated soil. From the batch chelant extraction studies, ethylenediaminetetraacetic acid (EDTA), citric acid, and nitrilotriacetic acid (NTA) were all effective in removing copper, lead, and zinc from the J-Field soils. Due to NTA being a Class II carcinogen, it is not recommended for use in remediating contaminated soils. EDTA and citric acid appear to offer the greatest potential as chelating agents to use in soil washing the Aberdeen Proving Ground soils. The other chelating agents studied (gluconate, oxalate, Citranox, ammonium acetate, and phosphoric acid, along with pH-adjusted water) were generally ineffective in mobilizing the heavy metals from the soils. The chelant solution removes the heavy metals (Cd, Cu, Pb, Zn, Fe, Cr, As, and Hg) simultaneously. Using a multiple-stage batch extraction

  15. General well function for soil vapor extraction

    Science.gov (United States)

    Perina, Tomas

    2014-04-01

    This paper develops a well function applicable to extraction of groundwater or soil vapor from a well under the most common field test conditions. The general well function (Perina and Lee, 2006) [12] is adapted to soil vapor extraction and constant head boundary at the top. For groundwater flow, the general well function now applies to an extraction well of finite diameter with uniform drawdown along the screen, finite-thickness skin, and partially penetrating an unconfined, confined, and leaky aquifer, or an aquifer underneath a reservoir. With a change of arguments, the model applies to soil vapor extraction from a vadose zone with no cover or with leaky cover at the ground surface. The extraction well can operate in specified drawdown (pressure for soil vapor) or specified flowrate mode. Frictional well loss is computed as flow-only dependent component of the drawdown inside the extraction well. In general case, the calculated flow distribution is not proportional to screen length for a multiscreen well.

  16. Impacts of Sampling and Handling Procedures on DNA- and RNA-based Microbial Characterization and Quantification of Groundwater and Saturated Soil

    Science.gov (United States)

    2012-07-01

    Interference of soil extracts and of the matrix itself (e.g., sand, clay, compost , activated carbon) to the derived DNA quantity and quality is often...phase. Both cultures were washed twice in sodium phosphate buffer, combined and inoculated to 100 g of homogenized soil at a moisture content

  17. Genomic DNA extraction from sapwood of Pinus roxburghii for ...

    African Journals Online (AJOL)

    A method for extraction of genomic DNA from sapwood tissues of mature tall trees of Pinus roxburghii, where collection of needle tissues is extremely difficult has been standardized. The extracted DNA was comparable to that obtained from the needle tissue in terms of yield and purity. The yield of extracted DNA ranged ...

  18. An efficient method for DNA extraction from Cladosporioid fungi

    OpenAIRE

    Moslem, M.A.; Bahkali, A.H.; Abd-Elsalam, K.A.; Wit, de, P.J.G.M.

    2010-01-01

    We developed an efficient method for DNA extraction from Cladosporioid fungi, which are important fungal plant pathogens. The cell wall of Cladosporioid fungi is often melanized, which makes it difficult to extract DNA from their cells. In order to overcome this we grew these fungi for three days on agar plates and extracted DNA from mycelium mats after manual or electric homogenization. High-quality DNA was isolated, with an A260/A280 ratio ranging between 1.6 and 2.0. Isolated genomic DNA w...

  19. Not all are free-living: high-throughput DNA metabarcoding reveals a diverse community of protists parasitizing soil metazoa.

    Science.gov (United States)

    Geisen, S; Laros, I; Vizcaíno, A; Bonkowski, M; de Groot, G A

    2015-09-01

    Protists, the most diverse eukaryotes, are largely considered to be free-living bacterivores, but vast numbers of taxa are known to parasitize plants or animals. High-throughput sequencing (HTS) approaches now commonly replace cultivation-based approaches in studying soil protists, but insights into common biases associated with this method are limited to aquatic taxa and samples. We created a mock community of common free-living soil protists (amoebae, flagellates, ciliates), extracted DNA and amplified it in the presence of metazoan DNA using 454 HTS. We aimed at evaluating whether HTS quantitatively reveals true relative abundances of soil protists and at investigating whether the expected protist community structure is altered by the co-amplification of metazoan-associated protist taxa. Indeed, HTS revealed fundamentally different protist communities from those expected. Ciliate sequences were highly over-represented, while those of most amoebae and flagellates were under-represented or totally absent. These results underpin the biases introduced by HTS that prevent reliable quantitative estimations of free-living protist communities. Furthermore, we detected a wide range of nonadded protist taxa probably introduced along with metazoan DNA, which altered the protist community structure. Among those, 20 taxa most closely resembled parasitic, often pathogenic taxa. Therewith, we provide the first HTS data in support of classical observational studies that showed that potential protist parasites are hosted by soil metazoa. Taken together, profound differences in amplification success between protist taxa and an inevitable co-extraction of protist taxa parasitizing soil metazoa obscure the true diversity of free-living soil protist communities. © 2015 John Wiley & Sons Ltd.

  20. Remediation of flare pit soils using supercritical fluid extraction

    Energy Technology Data Exchange (ETDEWEB)

    Nagpal, V.; Guigard, S.E. [Alberta Univ., Edmonton, AB (Canada). Dept. of Civil Engineering

    2005-09-01

    A laboratory study was conducted to examine the ability of supercritical fluid extraction (SFE) to remove petroleum hydrocarbons (PHCs) from two flare pit soils in Alberta. SFE is a technology for remediation of contaminated soils. In order to determine the optimal extraction conditions and to understand the effects of pressure, temperature, supercritical carbon dioxide flow rate, soil type, and extraction time on the extraction efficiency of SFE, extractions were performed on two flare pit soils at various pressures and temperatures. Chemicals in the study included diesel oil, SAE 10-30W motor oil, n-decane, hexadecane, tetratriacontane and pentacontane. The best extraction conditions were defined as conditions that result in a treated soil with a PHC concentration that meets the regulatory guidelines of the Canadian Council of Ministers of the Environment in the Canada-wide standard for PHC is soil. The study results indicate that the efficiency of the SFE process is solvent-density dependent for the conditions studied. The highest extraction efficiency for both soils was obtained at conditions of 24.1 MPa and 40 degrees C. An increase in pressure at a fixed temperature led to an increase in the extraction efficiency while an increase in temperature at a fixed pressure led to a decrease in the extraction efficiency. The treated soils were observed to be lighter in colour, drier, and grainier than the soil prior to extraction. It was concluded that SFE is an effective method for remediating flare pit soils. 63 refs., 4 tabs., 5 figs.

  1. Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise.

    Science.gov (United States)

    Soliman, Taha; Yang, Sung-Yin; Yamazaki, Tomoko; Jenke-Kodama, Holger

    2017-01-01

    Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil ® DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin ® Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA; P  technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

  2. Improved detection of endoparasite DNA in soil sample PCR by the use of anti-inhibitory substances.

    Science.gov (United States)

    Krämer, F; Vollrath, T; Schnieder, T; Epe, C

    2002-09-26

    Although there have been numerous microbial examinations of soil for the presence of human pathogenic developmental parasite stages of Ancylostoma caninum and Toxocara canis, molecular techniques (e.g. DNA extraction, purification and subsequent PCR) have scarcely been applied. Here, DNA preparations of soil samples artificially contaminated with genomic DNA or parasite eggs were examined by PCR. A. caninum and T. canis-specific primers based on the ITS-2 sequence were used for amplification. After the sheer DNA preparation a high content of PCR-interfering substances was still detectable. Subsequently, two different inhibitors of PCR-interfering agents (GeneReleaser, Bioventures Inc. and Maximator, Connex GmbH) were compared in PCR. Both substances increased PCR sensitivity greatly. However, comparison of the increase in sensitivity achieved with the two compounds demonstrated the superiority of Maximator, which enhanced sensitivity to the point of permitting positive detection of a single A. caninum egg and three T. canis eggs in a soil sample. This degree of sensitivity could not be achieved with GeneReleaser for either parasite Furthermore, Maximator not only increased sensitivity; it also cost less, required less time and had a lower risk of contamination. Future applications of molecular methods in epidemiological examinations of soil samples are discussed/elaborated.

  3. Influence of extractable soil manganese on oxidation capacity of different soils in Korea

    Science.gov (United States)

    Chon, Chul-Min; Kim, Jae Gon; Lee, Gyoo Ho; Kim, Tack Hyun

    2008-08-01

    We examined the relationship between soil oxidation capacity and extractable soil manganese, iron oxides, and other soil properties. The Korean soils examined in this study exhibited low to medium Cr oxidation capacities, oxidizing 0.00-0.47 mmol/kg, except for TG-4 soils, which had the highest capacity for oxidizing added Cr(III) [>1.01 mmol/kg of oxidized Cr(VI)]. TG and US soils, with high Mn contents, had relatively high oxidation capacities. The Mn amounts extracted by dithionite-citrate-bicarbonate (DCB) (Mnd), NH2OH·HCl (Mnh), and hydroquinone (Mnr) were generally very similar, except for the YS1 soils, and were well correlated. Only small proportions of either total Mn or DCB-extractable Mn were extracted by NH2OH·HCl and hydroquinone in the YS1 soils, suggesting inclusion of NH2OH·HCl and hydroquinone-resistant Mn oxides, because these extractants are weaker reductants than DCB. No Cr oxidation test results were closely related to total Mn concentrations, but Mnd, Mnh, and Mnr showed a relatively high correlation with the Cr tests ( r = 0.655-0.851; P Mnh were better correlated with the Cr oxidation tests than was the Mnr concentration, suggesting that the oxidation capacity of our soil samples can be better explained by Mnd and Mnh than by Mnr. The first component in principal components analysis indicated that extractable soil Mn was a main factor controlling net Cr oxidation in the soils. Total soil Mn, Fe oxides, and the clay fraction are crucial for predicting the mobility of pollutants and heavy metals in soils. The second principal component indicated that the presence of Fe oxides in soils had a significant relationship with the clay fraction and total Mn oxide, and was also related to heavy-metal concentrations (Zn, Cd, and Cu, but not Pb).

  4. An efficient method for DNA extraction from Cladosporioid fungi.

    Science.gov (United States)

    Moslem, M A; Bahkali, A H; Abd-Elsalam, K A; Wit, P J G M

    2010-11-23

    We developed an efficient method for DNA extraction from Cladosporioid fungi, which are important fungal plant pathogens. The cell wall of Cladosporioid fungi is often melanized, which makes it difficult to extract DNA from their cells. In order to overcome this we grew these fungi for three days on agar plates and extracted DNA from mycelium mats after manual or electric homogenization. High-quality DNA was isolated, with an A(260)/A(280) ratio ranging between 1.6 and 2.0. Isolated genomic DNA was efficiently digested with restriction enzymes and produced distinct banding patterns on agarose gels for the different Cladosporium species. Clear DNA fragments from the isolated DNA were amplified by PCR using small and large subunit rDNA primers, demonstrating that this method provides DNA of sufficiently high quality for molecular analyses.

  5. Extraction of DNA from plant and fungus tissues in situ

    Directory of Open Access Journals (Sweden)

    Abu Almakarem Amal S

    2012-06-01

    Full Text Available Abstract Background When samples are collected in the field and transported to the lab, degradation of the nucleic acids contained in the samples is frequently observed. Immediate extraction and precipitation of the nucleic acids reduces degradation to a minimum, thus preserving accurate sequence information. An extraction method to obtain high quality DNA in field studies is described. Findings DNA extracted immediately after sampling was compared to DNA extracted after allowing the sampled tissues to air dry at 21°C for 48 or 72 hours. While DNA extracted from fresh tissues exhibited little degradation, DNA extracted from all tissues exposed to 21°C air for 48 or 72 hours exhibited varying degrees of degradation. Yield was higher for extractions from fresh tissues in most cases. Four microcentrifuges were compared for DNA yield: one standard electric laboratory microcentrifuge (max rcf = 16,000×g, two battery-operated microcentrifuges (max rcf = 5,000 and 3,000 ×g, and one manually-operated microcentrifuge (max rcf = 120×g. Yields for all centrifuges were similar. DNA extracted under simulated field conditions was similar in yield and quality to DNA extracted in the laboratory using the same equipment. Conclusions This CTAB (cetyltrimethylammonium bromide DNA extraction method employs battery-operated and manually-operated equipment to isolate high quality DNA in the field. The method was tested on plant and fungus tissues, and may be adapted for other types of organisms. The method produced high quality DNA in laboratory tests and under simulated field conditions. The field extraction method should prove useful for working in remote sites, where ice, dry ice, and liquid nitrogen are unavailable; where degradation is likely to occur due to the long distances between the sample site and the laboratory; and in instances where other DNA preservation and transportation methods have been unsuccessful. It may be possible to adapt

  6. Microbial diversity in fecal samples depends on DNA extraction method

    DEFF Research Database (Denmark)

    Mirsepasi, Hengameh; Persson, Søren; Struve, Carsten

    2014-01-01

    was to evaluate two different DNA extraction methods in order to choose the most efficient method for studying intestinal bacterial diversity using Denaturing Gradient Gel Electrophoresis (DGGE). FINDINGS: In this study, a semi-automatic DNA extraction system (easyMag®, BioMérieux, Marcy I'Etoile, France......BACKGROUND: There are challenges, when extracting bacterial DNA from specimens for molecular diagnostics, since fecal samples also contain DNA from human cells and many different substances derived from food, cell residues and medication that can inhibit downstream PCR. The purpose of the study...... by easyMag® from the same fecal samples. Furthermore, DNA extracts obtained using easyMag® seemed to contain inhibitory compounds, since in order to perform a successful PCR-analysis, the sample should be diluted at least 10 times. DGGE performed on PCR from DNA extracted by QIAamp DNA Stool Mini Kit DNA...

  7. Mobility of arsenic and its compounds in soil and soil solution: the effect of soil pretreatment and extraction methods.

    Science.gov (United States)

    Száková, J; Tlustos, P; Goessler, W; Frková, Z; Najmanová, J

    2009-12-30

    The effect of soil extraction procedures and/or sample pretreatment (drying, freezing of the soil sample) on the extractability of arsenic and its compounds was tested. In the first part, five extraction procedures were compared with following order of extractable arsenic portions: 2M HNO(3)>0.43 M CH(3)COOH>or=0.05 M EDTA>or=Mehlich III (0.2M CH(3)COOH+0.25 M NH(4)NO(3)+0.013 M HNO(3)+0.015 M NH(4)F+0.001 M EDTA) extraction>water). Additionally, two methods of soil solution sampling were compared, centrifugation of saturated soil and the use of suction cups. The results showed that different sample pretreatments including soil solution sampling could lead to different absolute values of mobile arsenic content in soils. However, the interpretation of the data can lead to similar conclusions as apparent from the comparison of the soil solution sampling methods (r=0.79). For determination of arsenic compounds mild extraction procedures (0.05 M (NH(4))(2)SO(4), 0.01 M CaCl(2), and water) and soil solution sampling using suction cups were compared. Regarding the real soil conditions the extraction of fresh samples and/or in situ collection of soil solution are preferred among the sample pretreatments and/or soil extraction procedures. However, chemical stabilization of the solutions should be allowed and included in the analytical procedures for determination of individual arsenic compounds.

  8. Six-phase soil heating accelerates VOC extraction from clay soil

    International Nuclear Information System (INIS)

    Gauglitz, P.A.; Roberts, J.S.; Bergsman, T.M.; Caley, S.M.; Heath, W.O.; Miller, M.C.; Moss, R.W.; Schalla, R.; Jarosch, T.R.; Eddy-Dilek, C.A.

    1994-08-01

    Six-Phase Soil Heating (SPSH) was demonstrated as a viable technology for heating low permeability soils containing volatile organic contaminants. Testing was performed as part of the Volatile Organic Compounds in Non-Arid Soils Integrated Demonstration (VOC Non-Arid ID) at the Savannah River Site. The soil at the integrated demonstration site is contaminated with perchloroethylene (PCE) and trichloroethylene (TCE); the highest soil contamination occurs in clay-rich zones that are ineffectively treated by conventional soil vapor extraction due to the very low permeability of the clay. The SPSH demonstration sought to heat the clay zone and enhance the performance of conventional soil vapor extraction. Thermocouples at thirty locations quantified the areal and vertical heating within the treated zone. Soil samples were collected before and after heating to quantify the efficacy of heat-enhanced vapor extraction of PCE and TCE from the clay soil. Samples were taken (essentially every foot) from six wells prior to heating and adjacent to these wells after heating. Results show that contaminant removal from the clay zone was 99.7% (median) within the electrode array. Outside the array where the soil was heated, but to only 50 degrees C, the removal efficiency was 93%, showing that heating accelerated the removal of VOCs from the clay soil. The accelerated remediation resulted from effective heating of the contaminated clay zone by SPSH. The temperature of the clay zone increased to 100 degrees C after 8 days of heating and was maintained near 100 degrees C for 17 days. Electrical heating removed 19,000 gal of water from the soil as steam, with peak removal rate of 1,500 gpd of condensed steam

  9. Microbes on building materials — Evaluation of DNA extraction protocols as common basis for molecular analysis

    International Nuclear Information System (INIS)

    Ettenauer, Jörg D.; Piñar, Guadalupe; Lopandic, Ksenija; Spangl, Bernhard; Ellersdorfer, Günther; Voitl, Christian; Sterflinger, Katja

    2012-01-01

    The study of microbial life in building materials is an emerging topic concerning biodeterioration of materials as well as health risks in houses and at working places. Biodegradation and potential health implications associated with microbial growth in our residues claim for more precise methods for quantification and identification. To date, cultivation experiments are commonly used to gain insight into the microbial diversity. Nowadays, molecular techniques for the identification of microorganisms provide efficient methods that can be applied in this field. The efficiency of DNA extraction is decisive in order to perform a reliable and reproducible quantification of the microorganisms by qPCR or to characterize the structure of the microbial community. In this study we tested thirteen DNA extraction methods and evaluated their efficiency for identifying (1) the quantity of DNA, (2) the quality and purity of DNA and (3) the ability of the DNA to be amplified in a PCR reaction using three universal primer sets for the ITS region of fungi as well as one primer pair targeting the 16S rRNA of bacteria with three typical building materials — common plaster, red brick and gypsum cardboard. DNA concentration measurements showed strong variations among the tested methods and materials. Measurement of the DNA yield showed up to three orders of magnitude variation from the same samples, whereas A260/A280 ratios often prognosticated biases in the PCR amplifications. Visualization of the crude DNA extracts and the comparison of DGGE fingerprints showed additional drawbacks of some methods. The FastDNA Spin kit for soil showed to be the best DNA extraction method and could provide positive results for all tests with the three building materials. Therefore, we suggest this method as a gold standard for quantification of indoor fungi and bacteria in building materials. -- Highlights: ► Up to thirteen extraction methods were evaluated with three building materials.

  10. Case study of shallow soil mixing and soil vacuum extraction remediation project

    International Nuclear Information System (INIS)

    Carey, M.J.; Day, S.R.; Pinewski, R.; Schroder, D.

    1995-01-01

    Shallow Soil Mixing (SSM) and Soil Vacuum Extraction (SVE) are techniques which have been increasingly relied on for the insitu remediation of contaminated soils. The primary applications of SSM have been to mix cement, bentonite, or other reagents to modify properties and thereby remediate contaminated soils or sludges. Soil vacuum extraction has been used at numerous applications for insitu removal of contaminants from soils. At a recent project in southern Ohio, the two technologies were integrated and enhanced to extract volatile organic compounds (VOCs) from soils at a Department of Energy facility. Advantages of the integrated SSM/SVE technology over alternative technologies include a relatively rapid remediation compared to other in-situ techniques at a lower cost, less exposure of waste to the surface environment and elimination of off-site disposal. These advantages led to the selection of the use of both technologies on the project in Southern Ohio. The information presented in this paper is intended to provide Engineers and owners with the level of understanding necessary to apply soil mixing and vacuum extraction technology to a specific site. The most important steps in implementing the technology are site investigation, feasibility estimate, selection of performance criteria, selection of appropriate materials, bench scale testing and construction

  11. Evaluation of four automated protocols for extraction of DNA from FTA cards.

    Science.gov (United States)

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura; Frank-Hansen, Rune; Poulsen, Lena; Hansen, Anders J; Morling, Niels

    2013-10-01

    Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore, we demonstrated that it was possible to successfully extract sufficient DNA for STR profiling from previously processed FTA card pieces that had been stored at 4 °C for up to 1 year. This showed that rare or precious FTA card samples may be saved for future analyses even though some DNA was already extracted from the FTA cards.

  12. Hanford soil partitioning and vapor extraction study

    International Nuclear Information System (INIS)

    Yonge, D.; Hossain, A.; Cameron, R.; Ford, H.; Storey, C.

    1996-07-01

    This report describes the testing and results of laboratory experiments conducted to assist the carbon tetrachloride soil vapor extraction project operating in the 200 West Area of the Hanford Site in Richland, Washington. Vapor-phase adsorption and desorption testing was performed using carbon tetrachloride and Hanford Site soils to estimate vapor-soil partitioning and reasonably achievable carbon tetrachloride soil concentrations during active vapor extractions efforts at the 200 West Area. (CCl 4 is used in Pu recovery from aqueous streams.)

  13. Microbes on building materials - Evaluation of DNA extraction protocols as common basis for molecular analysis

    Energy Technology Data Exchange (ETDEWEB)

    Ettenauer, Joerg D., E-mail: joerg.ettenauer@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria); Pinar, Guadalupe, E-mail: Guadalupe.Pinar@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria); Lopandic, Ksenija, E-mail: Ksenija.Lopandic@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria); Spangl, Bernhard, E-mail: Bernhard.Spangl@boku.ac.at [University of Natural Resources and Life Sciences, Department of Landscape, Spatial and Infrastructure Science, Institute of Applied Statistics and Computing (IASC), Gregor Mendel-Str. 33, A-1180 Vienna (Austria); Ellersdorfer, Guenther, E-mail: Guenther.Ellersdorfer@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria); Voitl, Christian, E-mail: Christian.Voitl@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria); Sterflinger, Katja, E-mail: Katja.Sterflinger@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria)

    2012-11-15

    The study of microbial life in building materials is an emerging topic concerning biodeterioration of materials as well as health risks in houses and at working places. Biodegradation and potential health implications associated with microbial growth in our residues claim for more precise methods for quantification and identification. To date, cultivation experiments are commonly used to gain insight into the microbial diversity. Nowadays, molecular techniques for the identification of microorganisms provide efficient methods that can be applied in this field. The efficiency of DNA extraction is decisive in order to perform a reliable and reproducible quantification of the microorganisms by qPCR or to characterize the structure of the microbial community. In this study we tested thirteen DNA extraction methods and evaluated their efficiency for identifying (1) the quantity of DNA, (2) the quality and purity of DNA and (3) the ability of the DNA to be amplified in a PCR reaction using three universal primer sets for the ITS region of fungi as well as one primer pair targeting the 16S rRNA of bacteria with three typical building materials - common plaster, red brick and gypsum cardboard. DNA concentration measurements showed strong variations among the tested methods and materials. Measurement of the DNA yield showed up to three orders of magnitude variation from the same samples, whereas A260/A280 ratios often prognosticated biases in the PCR amplifications. Visualization of the crude DNA extracts and the comparison of DGGE fingerprints showed additional drawbacks of some methods. The FastDNA Spin kit for soil showed to be the best DNA extraction method and could provide positive results for all tests with the three building materials. Therefore, we suggest this method as a gold standard for quantification of indoor fungi and bacteria in building materials. -- Highlights: Black-Right-Pointing-Pointer Up to thirteen extraction methods were evaluated with three

  14. Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise

    Directory of Open Access Journals (Sweden)

    Taha Soliman

    2017-12-01

    Full Text Available Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T using two different DNA extraction kits: (1 MO BIO PowerSoil® DNA Isolation kit (MO_R and MO_T and (2 NucleoSpin® Soil kit (MN_R and MN_T. Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes, obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA; P < 0.006. In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

  15. Detection of DNA specific sequences of Spongospora subterranea in soil and potato tubers

    Directory of Open Access Journals (Sweden)

    Cristian Oswaldo Saavedra Rodríguez

    2004-01-01

    Full Text Available A test has been developed for early identification of the casual agent for potato powdery scab (Spongospora subterranea fs subterranea. Identification was carried out in seeds and soil where this tuber is grown. A polymerase chain reaction (PCR was set up for detecting 372, 390 and 391 bp ribosomal DNA internal transcribed spacer sequences (ITS in the S. subterraneagenome. A method for extracting and purifying DNA from infected plant material (potato root nodes and pustules on the potato was standardised. Plant tissue was obtained by potato tuber propagation using an inoculum from the pathogen in greenhouse conditions. After the PCR had been optimised and its sensitivity determined, a molecular methodology was validated by examining plant material infected with S. subterranea and soil samples infested with the pathogen obtained from the departments of Cundinamarca and Nariño. The PCR detected S. subterranea DNA from infected material and soil samples (all thirty samples from the experimental area analysed proved PCR positive. These results show that this molecular method was not just useful for the early detection of the pathogen in soil samples but as a tool for detecting or determining the possible presence of this micro-organism in places that have been declared f ree of S. subterranea and an effective form of quality control in producing the certif ied potato seed. Key words: Powdery scab, cystosori, internal transcribed spacer, PCR, plasmodiophorid.

  16. DNA extraction method for PCR in mycorrhizal fungi.

    Science.gov (United States)

    Manian, S; Sreenivasaprasad, S; Mills, P R

    2001-10-01

    To develop a simple and rapid DNA extraction protocol for PCR in mycorrhizal fungi. The protocol combines the application of rapid freezing and boiling cycles and passage of the extracts through DNA purification columns. PCR amplifiable DNA was obtained from a number of endo- and ecto-mycorrhizal fungi using minute quantities of spores and mycelium, respectively. DNA extracted following the method, was used to successfully amplify regions of interest from high as well as low copy number genes. The amplicons were suitable for further downstream applications such as sequencing and PCR-RFLPs. The protocol described is simple, short and facilitates rapid isolation of PCR amplifiable genomic DNA from a large number of fungal isolates in a single day. The method requires only minute quantities of starting material and is suitable for mycorrhizal fungi as well as a range of other fungi.

  17. Development of an extraction method for perchlorate in soils.

    Science.gov (United States)

    Cañas, Jaclyn E; Patel, Rashila; Tian, Kang; Anderson, Todd A

    2006-03-01

    Perchlorate originates as a contaminant in the environment from its use in solid rocket fuels and munitions. The current US EPA methods for perchlorate determination via ion chromatography using conductivity detection do not include recommendations for the extraction of perchlorate from soil. This study evaluated and identified appropriate conditions for the extraction of perchlorate from clay loam, loamy sand, and sandy soils. Based on the results of this evaluation, soils should be extracted in a dry, ground (mortar and pestle) state with Milli-Q water in a 1 ratio 1 soil ratio water ratio and diluted no more than 5-fold before analysis. When sandy soils were extracted in this manner, the calculated method detection limit was 3.5 microg kg(-1). The findings of this study have aided in the establishment of a standardized extraction method for perchlorate in soil.

  18. Predicting Soil-Air and Soil-Water Transport Properties During Soil Vapor Extraction

    DEFF Research Database (Denmark)

    Poulsen, Tjalfe

    Increased application of in-situ technology for control and removal of volatile organic compounds (VOC) in the subsurface has made the understanding of soil physical properties and their impact upon contaminant transport even more important. Knowledge of contaminant transport is important when...... properties of undisturbed soil from more easily measurable soil properties are developed. The importance of soil properties with respect to contaminant migration during remediation by soil vapor extraction (SVE) in the unsaturated zone was investigated using numerical simulations....

  19. DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

    International Nuclear Information System (INIS)

    Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D.

    1991-01-01

    Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links

  20. How Severely Is DNA Quantification Hampered by RNA Co-extraction?

    Science.gov (United States)

    Sanchez, Ignacio; Remm, Matthieu; Frasquilho, Sonia; Betsou, Fay; Mathieson, William

    2015-10-01

    The optional RNase digest that is part of many DNA extraction protocols is often omitted, either because RNase is not provided in the kit or because users do not want to risk contaminating their laboratory. Consequently, co-eluting RNA can become a "contaminant" of unknown magnitude in a DNA extraction. We extracted DNA from liver, lung, kidney, and heart tissues and established that 28-52% of the "DNA" as assessed by spectrophotometry is actually RNA (depending on tissue type). Including an RNase digest in the extraction protocol reduced 260:280 purity ratios. Co-eluting RNA drives an overestimation of DNA yield when quantification is carried out using OD 260 nm spectrophotometry, or becomes an unquantified contaminant when spectrofluorometry is used for DNA quantification. This situation is potentially incompatible with the best practice guidelines for biobanks issued by organizations such as the International Society for Biological and Environmental Repositories, which state that biospecimens should be accurately characterized in terms of their identity, purity, concentration, and integrity. Consequently, we conclude that an RNase digest must be included in DNA extractions if pure DNA is required. We also discuss the implications of unquantified RNA contamination in DNA samples in the context of laboratory accreditation schemes.

  1. Integrated vacuum extraction/pneumatic soil fracturing system for remediation of low permeability soil

    International Nuclear Information System (INIS)

    Plaines, A.L.; Piniewski, R.J.; Yarbrough, G.D.

    1994-01-01

    There is wide use of vacuum extraction to remove volatile and semi-volatile organic compounds (VOCs) from unsaturated soil. At sites with soil of low permeability, VOC extraction rates may not be sufficient to meet soil clean-up objectives within the desired time frame. During vacuum extraction in low permeability soil, the diffusion rates of VOCs through the soil matrix may limit VOC removal rates. An increase in the number of subsurface paths for advective flow through the contaminated zone results in a larger mass of contaminant being removed in a shorter time frame, accelerating site remediation. One technique for increasing the number of subsurface flow paths is Terra Vac's process of pneumatic soil fracturing (PSF). In this process, pressurized air is injected into the subsurface, creating micro-fractures for the vacuum extraction system to withdraw contaminants. Similar to hydraulic fracturing techniques long used in the petroleum industry for increasing yield from oil and gas production wells, this technique has applications for soil remediation in low permeability conditions. Two case studies, one in Louisiana at a gasoline service station and one at a manufacturing plant in New York, are presented

  2. DNA extraction from sea anemone (Cnidaria: Actiniaria tissues for molecular analyses

    Directory of Open Access Journals (Sweden)

    Pinto S.M.

    2000-01-01

    Full Text Available A specific DNA extraction method for sea anemones is described in which extraction of total DNA from eight species of sea anemones and one species of corallimorpharian was achieved by changing the standard extraction protocols. DNA extraction from sea anemone tissue is made more difficult both by the tissue consistency and the presence of symbiotic zooxanthellae. The technique described here is an efficient way to avoid problems of DNA contamination and obtain large amounts of purified and integral DNA which can be used in different kinds of molecular analyses.

  3. Supercritical Fluid Extraction of Plutonium and Americium from Soil

    International Nuclear Information System (INIS)

    Fox, R.V.; Mincher, B.J.

    2002-01-01

    Supercritical fluid extraction (SFE) of plutonium and americium from soil was successfully demonstrated using supercritical fluid carbon dioxide solvent augmented with organophosphorus and beta-diketone complexants. Spiked Idaho soils were chemically and radiologically characterized, then extracted with supercritical fluid carbon dioxide at 2,900 psi and 65 C containing varying concentrations of tributyl phosphate (TBP) and thenoyltrifluoroacetone (TTA). A single 45 minute SFE with 2.7 mol% TBP and 3.2 mol% TTA provided as much as 88% ± 6.0 extraction of americium and 69% ± 5.0 extraction of plutonium. Use of 5.3 mol% TBP with 6.8 mol% of the more acidic beta-diketone hexafluoroacetylacetone (HFA) provided 95% ± 3.0 extraction of americium and 83% ± 5.0 extraction of plutonium in a single 45 minute SFE at 3,750 psi and 95 C. Sequential chemical extraction techniques were used to chemically characterize soil partitioning of plutonium and americium in pre-SFE soil samples. Sequential chemical extraction techniques demonstrated that spiked plutonium resides primarily (76.6%) in the sesquioxide fraction with minor amounts being absorbed by the oxidizable fraction (10.6%) and residual fractions (12.8%). Post-SFE soils subjected to sequential chemical extraction characterization demonstrated that 97% of the oxidizable, 78% of the sesquioxide and 80% of the residual plutonium could be removed using SFE. These preliminary results show that SFE may be an effective solvent extraction technique for removal of actinide contaminants from soil

  4. Extraction of genomic DNA from Melipona quadrifasciata (Hymenoptera: Apidae, Meliponinae)

    OpenAIRE

    Waldschmidt, Ana Maria; Salomão, Tânia Maria Fernandes; Barros, Everaldo Gonçalves de; Campos, Lúcio de Antônio Oliveira

    1997-01-01

    The objective of the present study was to test three different procedures for DNA extraction of Melipona quadrifasciata based on existing methods for DNA extraction of Apis, plants and fungi. These methods differ in the concentrations of specific substances in the extraction buffer. The results demonstrate that the method used for Apis is not adequate for DNA extraction from M. quadrifasciata. On the other hand, with minor modifications this method and the methods for plants and fungi were ad...

  5. Extraction agents for the removal of polycyclic aromatic hydrocarbons (PAHs) from soil in soil washing technologies

    International Nuclear Information System (INIS)

    Lau, Ee Von; Gan, Suyin; Ng, Hoon Kiat; Poh, Phaik Eong

    2014-01-01

    Polycyclic aromatic hydrocarbons (PAHs) in soil have been recognised as a serious health and environmental issue due to their carcinogenic, mutagenic and teratogenic properties. One of the commonly employed soil remediation techniques to clean up such contamination is soil washing or solvent extraction. The main factor which governs the efficiency of this process is the solubility of PAHs in the extraction agent. Past field-scale soil washing treatments for PAH-contaminated soil have mainly employed organic solvents or water which is either toxic and costly or inefficient in removing higher molecular weight PAHs. Thus, the present article aims to provide a review and discussion of the alternative extraction agents that have been studied, including surfactants, biosurfactants, microemulsions, natural surfactants, cyclodextrins, vegetable oil and solution with solid phase particles. These extraction agents have been found to remove PAHs from soil at percentages ranging from 47 to 100% for various PAHs. -- Highlights: • The alternative and advancement in extraction agents to remove PAHs from soil using soil washing technology is summarised. • The soil regulations for PAH level in various countries are summarized for reference to researchers. • The concentration levels of PAHs in soil at present and the need for soil remediation is presented. -- The efficiency of the extraction agent plays a significant role in soil washing of PAH-contaminated soil

  6. Food Fish Identification from DNA Extraction through Sequence Analysis

    Science.gov (United States)

    Hallen-Adams, Heather E.

    2015-01-01

    This experiment exposed 3rd and 4th y undergraduates and graduate students taking a course in advanced food analysis to DNA extraction, polymerase chain reaction (PCR), and DNA sequence analysis. Students provided their own fish sample, purchased from local grocery stores, and the class as a whole extracted DNA, which was then subjected to PCR,…

  7. Evaluation of Four Automated Protocols for Extraction of DNA from FTA Cards

    OpenAIRE

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura; Frank-Hansen, Rune; Poulsen, Lena; Hansen, Anders J; Morling, Niels

    2013-01-01

    Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cell...

  8. Extraction of Plutonium From Spiked INEEL Soil Samples Using the Ligand-Assisted Supercritical Fluid Extraction (LA-SFE) Technique

    International Nuclear Information System (INIS)

    Fox, R.V.; Mincher, B.J.; Holmes, R.G.G.

    1999-01-01

    In order to investigate the effectiveness of ligand-assisted supercritical fluid extraction for the removal of transuranic contaminations from soils an Idaho National Engineering and Environmental Laboratory (INEEL) silty-clay soil sample was obtained from near the Radioactive Waste Management Complex area and subjected to three different chemical preparations before being spiked with plutonium. The spiked INEEL soil samples were subjected to a sequential aqueous extraction procedure to determine radionuclide portioning in each sample. Results from those extractions demonstrate that plutonium consistently partitioned into the residual fraction across all three INEEL soil preparations whereas americium partitioned 73% into the iron/manganese fraction for soil preparation A, with the balance partitioning into the residual fraction. Plutonium and americium were extracted from the INEEL soil samples using a ligand-assisted supercritical fluid extraction technique. Initial supercritical fluid extraction runs produced plutonium extraction technique. Initial supercritical fluid extraction runs produced plutonium extraction efficiencies ranging from 14% to 19%. After a second round wherein the initial extraction parameters were changed, the plutonium extraction efficiencies increased to 60% and as high as 80% with the americium level in the post-extracted soil samples dropping near to the detection limits. The third round of experiments are currently underway. These results demonstrate that the ligand-assisted supercritical fluid extraction technique can effectively extract plutonium from the spiked INEEL soil preparations

  9. Modified DNA extraction for rapid PCR detection of methicillin-resistant staphylococci

    International Nuclear Information System (INIS)

    Japoni, A.; Alborzi, A.; Rasouli, M.; Pourabbas, B.

    2004-01-01

    Nosocomial infection caused by methicillin-resistant staphylococci poses a serious problem in many countries. The aim of this study was to rapidly and reliably detect methicillin-resistant-staphylococci in order to suggest appropriate therapy. The presence or absence of the methicillin-resistance gene in 115 clinical isolates of staphylococcus aureus and 50 isolates of coagulase negative staphylococci was examined by normal PCR. DNA extraction for PCR performance was then modified by omission of achromopeptadiase and proteinase K digestion, phenol/chloroform extraction and ethanol precipitation. All isolates with Mic>8 μ g/ml showed positive PCR. No differences in PCR detection have been observed when normal and modified DNA extractions have been performed. Our modified DNA extraction can quickly detect methicillin-resistant staphylococci by PCR. The advantage of rapid DNA extraction extends to both reduction of time and cost of PCR performance. This modified DNA extraction is suitable for different PCR detection, when staphylococci are the subject of DNA analysis

  10. Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

    Science.gov (United States)

    Makarova, Alena V; Grabow, Corinn; Gening, Leonid V; Tarantul, Vyacheslav Z; Tahirov, Tahir H; Bessho, Tadayoshi; Pavlov, Youri I

    2011-01-31

    Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+) ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA"). We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

  11. Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

    Directory of Open Access Journals (Sweden)

    Alena V Makarova

    2011-01-01

    Full Text Available Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+ ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA". We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

  12. [DNA Extraction from Old Bones by AutoMate Express™ System].

    Science.gov (United States)

    Li, B; Lü, Z

    2017-08-01

    To establish a method for extracting DNA from old bones by AutoMate Express™ system. Bones were grinded into powder by freeze-mill. After extraction by AutoMate Express™, DNA were amplified and genotyped by Identifiler®Plus and MinFiler™ kits. DNA were extracted from 10 old bone samples, which kept in different environments with the postmortem interval from 10 to 20 years, in 3 hours by AutoMate Express™ system. Complete STR typing results were obtained from 8 samples. AutoMate Express™ system can quickly and efficiently extract DNA from old bones, which can be applied in forensic practice. Copyright© by the Editorial Department of Journal of Forensic Medicine

  13. Soil solution extraction techniques for microbial ecotoxicity testing: a comparative evaluation.

    Science.gov (United States)

    Tiensing, T; Preston, S; Strachan, N; Paton, G I

    2001-02-01

    The suitability of two different techniques (centrifugation and Rhizon sampler) for obtaining the interstitial pore water of soil (soil solution), integral to the ecotoxicity assessment of metal contaminated soil, were investigated by combining chemical analyses and a luminescence-based microbial biosensor. Two different techniques, centrifugation and Rhizon sampler, were used to extract the soil solution from Insch (a loamy sand) and Boyndie (a sandy loam) soils, which had been amended with different concentrations of Zn and Cd. The concentrations of dissolved organic carbon (DOC), major anions (F- , CI-, NO3, SO4(2-)) and major cations (K+, Mg2+, Ca2+) in the soil solutions varied depending on the extraction technique used. Overall, the concentrations of Zn and Cd were significantly higher in the soil solution extracted using the centrifugation technique compared with that extracted using the Rhizon sampler technique. Furthermore, the differences observed between the two extraction techniques depended on the type of soil from which the solution was being extracted. The luminescence-based biosensor Escherichia coli HB101 pUCD607 was shown to respond to the free metal concentrations in the soil solutions and showed that different toxicities were associated with each soil, depending on the technique used to extract the soil solution. This study highlights the need to characterise the type of extraction technique used to obtain the soil solution for ecotoxicity testing in order that a representative ecotoxicity assessment can be carried out.

  14. Filter Membrane Effects on Water-Extractable Phosphorus Concentrations from Soil.

    Science.gov (United States)

    Norby, Jessica; Strawn, Daniel; Brooks, Erin

    2018-03-01

    To accurately assess P concentrations in soil extracts, standard laboratory practices for monitoring P concentrations are needed. Water-extractable P is a common analytical test to determine P availability for leaching from soils, and it is used to determine best management practices. Most P analytical tests require filtration through a filter membrane with 0.45-μm pore size to distinguish between particulate and dissolved P species. However, filter membrane type is rarely specified in method protocols, and many different types of membranes are available. In this study, three common filter membrane materials (polyether sulfone, nylon, and nitrocellulose), all with 0.45-μm pore sizes, were tested for analytical differences in total P concentrations and dissolved reactive P (DRP) concentrations in water extracts from six soils sampled from two regions. Three of the extracts from the six soil samples had different total P concentrations for all three membrane types. The other three soil extracts had significantly different total P results from at least one filter membrane type. Total P concentration differences were as great as 35%. The DRP concentrations in the extracts were dependent on filter type in five of the six soil types. Results from this research show that filter membrane type is an important parameter that affects concentrations of total P and DRP from soil extracts. Thus, membrane type should be specified in soil extraction protocols. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

  15. Rapid DNA extraction of bacterial genome using laundry detergents ...

    African Journals Online (AJOL)

    Genomic DNA extraction from bacterial cells involves processes normally performed in most biological laboratories. Therefore, various methods have been offered, manually and kit, but these methods may be time consuming and costly. In this paper, genomic DNA extraction of Pseudomonas aeruginosa was investigated ...

  16. Rapid DNA extraction of bacterial genome using laundry detergents ...

    African Journals Online (AJOL)

    Yomi

    2012-01-03

    Jan 3, 2012 ... Genomic DNA extraction from bacterial cells involves processes normally performed in most biological laboratories. Therefore, various methods have been offered, manually and kit, but these methods may be time consuming and costly. In this paper, genomic DNA extraction of Pseudomonas aeruginosa ...

  17. Highly efficient DNA extraction method from skeletal remains

    Directory of Open Access Journals (Sweden)

    Irena Zupanič Pajnič

    2011-03-01

    Full Text Available Background: This paper precisely describes the method of DNA extraction developed to acquire high quality DNA from the Second World War skeletal remains. The same method is also used for molecular genetic identification of unknown decomposed bodies in routine forensic casework where only bones and teeth are suitable for DNA typing. We analysed 109 bones and two teeth from WWII mass graves in Slovenia. Methods: We cleaned the bones and teeth, removed surface contaminants and ground the bones into powder, using liquid nitrogen . Prior to isolating the DNA in parallel using the BioRobot EZ1 (Qiagen, the powder was decalcified for three days. The nuclear DNA of the samples were quantified by real-time PCR method. We acquired autosomal genetic profiles and Y-chromosome haplotypes of the bones and teeth with PCR amplification of microsatellites, and mtDNA haplotypes 99. For the purpose of traceability in the event of contamination, we prepared elimination data bases including genetic profiles of the nuclear and mtDNA of all persons who have been in touch with the skeletal remains in any way. Results: We extracted up to 55 ng DNA/g of the teeth, up to 100 ng DNA/g of the femurs, up to 30 ng DNA/g of the tibias and up to 0.5 ng DNA/g of the humerus. The typing of autosomal and YSTR loci was successful in all of the teeth, in 98 % dekalof the femurs, and in 75 % to 81 % of the tibias and humerus. The typing of mtDNA was successful in all of the teeth, and in 96 % to 98 % of the bones. Conclusions: We managed to obtain nuclear DNA for successful STR typing from skeletal remains that were over 60 years old . The method of DNA extraction described here has proved to be highly efficient. We obtained 0.8 to 100 ng DNA/g of teeth or bones and complete genetic profiles of autosomal DNA, Y-STR haplotypes, and mtDNA haplotypes from only 0.5g bone and teeth samples.

  18. DNA extraction methods for detecting genetically modified foods: A comparative study.

    Science.gov (United States)

    Elsanhoty, Rafaat M; Ramadan, Mohamed Fawzy; Jany, Klaus Dieter

    2011-06-15

    The work presented in this manuscript was achieved to compare six different methods for extracting DNA from raw maize and its derived products. The methods that gave higher yield and quality of DNA were chosen to detect the genetic modification in the samples collected from the Egyptian market. The different methods used were evaluated for extracting DNA from maize kernels (without treatment), maize flour (mechanical treatment), canned maize (sweet corn), frozen maize (sweet corn), maize starch, extruded maize, popcorn, corn flacks, maize snacks, and bread made from corn flour (mechanical and thermal treatments). The quality and quantity of the DNA extracted from the standards, containing known percentages of GMO material and from the different food products were evaluated. For qualitative detection of the GMO varieties in foods, the GMOScreen 35S/NOS test kit was used, to screen the genetic modification in the samples. The positive samples for the 35S promoter and/or the NOS terminator were identified by the standard methods adopted by EU. All of the used methods extracted yielded good DNA quality. However, we noted that the purest DNA extract were obtained using the DNA extraction kit (Roche) and this generally was the best method for extracting DNA from most of the maize-derived foods. We have noted that the yield of DNA extracted from maize-derived foods was generally lower in the processed products. The results indicated that 17 samples were positive for the presence of 35S promoter, while 34% from the samples were positive for the genetically modified maize line Bt-176. Copyright © 2010 Elsevier Ltd. All rights reserved.

  19. Comparison of Chemical Extraction Methods for Determination of Soil Potassium in Different Soil Types

    Science.gov (United States)

    Zebec, V.; Rastija, D.; Lončarić, Z.; Bensa, A.; Popović, B.; Ivezić, V.

    2017-12-01

    Determining potassium supply of soil plays an important role in intensive crop production, since it is the basis for balancing nutrients and issuing fertilizer recommendations for achieving high and stable yields within economic feasibility. The aim of this study was to compare the different extraction methods of soil potassium from arable horizon of different types of soils with ammonium lactate method (KAL), which is frequently used as analytical method for determining the accessibility of nutrients and it is a common method used for issuing fertilizer recommendations in many Europe countries. In addition to the ammonium lactate method (KAL, pH 3.75), potassium was extracted with ammonium acetate (KAA, pH 7), ammonium acetate ethylenediaminetetraacetic acid (KAAEDTA, pH 4.6), Bray (KBRAY, pH 2.6) and with barium chloride (K_{BaCl_2 }, pH 8.1). The analyzed soils were extremely heterogeneous with a wide range of determined values. Soil pH reaction ( {pH_{H_2 O} } ) ranged from 4.77 to 8.75, organic matter content ranged from 1.87 to 4.94% and clay content from 8.03 to 37.07%. In relation to KAL method as the standard method, K_{BaCl_2 } method extracts 12.9% more on average of soil potassium, while in relation to standard method, on average KAA extracts 5.3%, KAAEDTA 10.3%, and KBRAY 27.5% less of potassium. Comparison of analyzed extraction methods of potassium from the soil is of high precision, and most reliable comparison was KAL method with KAAEDTA, followed by a: KAA, K_{BaCl_2 } and KBRAY method. Extremely significant statistical correlation between different extractive methods for determining potassium in the soil indicates that any of the methods can be used to accurately predict the concentration of potassium in the soil, and that carried out research can be used to create prediction model for concentration of potassium based on different methods of extraction.

  20. SOIL VAPOR EXTRACTION TECHNOLOGY: REFERENCE HANDBOOK

    Science.gov (United States)

    Soil vapor extraction (SVE) systems are being used in Increasing numbers because of the many advantages these systems hold over other soil treatment technologies. SVE systems appear to be simple in design and operation, yet the fundamentals governing subsurface vapor transport ar...

  1. Molecular analysis of manufactured gas plant soils for naphthalene mineralization

    International Nuclear Information System (INIS)

    Sanseverino, J.; Werner, C.; Fleming, J.; Applegate, B.M.; King, J.M.H.; Sayler, G.S.; Blackburn, J.

    1991-01-01

    New molecular tools are being developed and tested to ascertain the biodegradability of hazardous wastes by soil bacterial population. The potential for manufactured gas plant (MGP) soil bacterial populations to degrade naphthalene, as a component mixture of polynuclear aromatic hydrocarbons, was evaluated by the detection of a naphthalene biodegradative genotype by DNA probe hybridization with DNA extracts and colonies of cultured bacteria of the MGP soils. The activity of the naphthalene-degrading populations was evaluated by mineralization assays, 14 CO 2 production from 14 C-naphthalene. Direct messenger RNA (mRNA) extraction from MGP soil was evaluated as an instantaneous measure of naphthalene catabolic gene expression in MGP soil. The bioavailability of naphthalene for bacterial degradation within the MGP soils was assessed by measuring the bioluminescent response of a naphthalene-lux catabolic reporter strain Pseudomonas fluorescens HK44 (pUTK21). DNA extracted from 5 MGP soils and 1 creosote-contaminated soil and hybridized with a nahA gene probe indicated that the naphthalene degradative genes were present in all samples in the range of 0.06 to 0.95 ng/100 μl DNA extract which was calculated to represent 3.58 x 10 8 to 1.05 x 10 10 nahA positive cells/g soil. Phenanthrene, anthracene, and benzo(a)pyrene were mineralized also by some of the soils. NAH7 homologous messenger RNA transcripts were detectable in one MGP soil and in the creosote-contaminated soil

  2. Evaluation of Four Automated Protocols for Extraction of DNA from FTA Cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura

    2013-01-01

    protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA...... from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore......, we demonstrated that it was possible to successfully extract sufficient DNA for STR profiling from previously processed FTA card pieces that had been stored at 4 °C for up to 1 year. This showed that rare or precious FTA card samples may be saved for future analyses even though some DNA was already...

  3. extraction of high quality dna from polysaccharides-secreting ...

    African Journals Online (AJOL)

    cistvr

    A DNA extraction method using CTAB was used for the isolation of genomic DNA from ten. Xanthomonas campestris pathovars, ten isolates of Xanthomonas albilineans and one isolate of. Pseudomonas rubrisubalbicans. High quality DNA was obtained that was ideal for molecular analy- ses. Extracellular polysaccharides ...

  4. Genomic DNA extraction method from Annona senegalensis Pers ...

    African Journals Online (AJOL)

    Extraction of DNA in many plants is difficult because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, replications, amplification, as well as cloning. Modified procedure based on the hexadecyltrimethyl ammoniumbromide (CTAB) method is ...

  5. Optimization of DNA extraction for ISSR studies in Tectona grandis Lf

    African Journals Online (AJOL)

    GRACE

    2006-07-03

    Jul 3, 2006 ... genomic DNA, emphasizing screening of inexpensive, rapid and simple DNA extraction methods (Weishing et al., 1995). Yield and quality of DNA often varies among tree tissue types (Henry, 2001). Besides, purification of genomic DNA in trees is difficult due to co-extraction of high quantities of tannins, ...

  6. Extraction of Chromosomal DNA from Schizosaccharomyces pombe.

    Science.gov (United States)

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-05-02

    Extraction of DNA from Schizosaccharomyces pombe cells is required for various uses, including templating polymerase chain reactions (PCRs), Southern blotting, library construction, and high-throughput sequencing. To purify high-quality DNA, the cell wall is removed by digestion with Zymolyase or Lyticase and the resulting spheroplasts lysed using sodium dodecyl sulfate (SDS). Cell debris, SDS, and SDS-protein complexes are subsequently precipitated by the addition of potassium acetate and removed by centrifugation. Finally, DNA is precipitated using isopropanol. At this stage, purity is usually sufficient for PCR. However, for more sensitive procedures, such as restriction enzyme digestion, additional purification steps, including proteinase K digestion and phenol-chloroform extraction, are recommended. All of these steps are described in detail here. © 2016 Cold Spring Harbor Laboratory Press.

  7. Genomic DNA extraction method from pearl millet ( Pennisetum ...

    African Journals Online (AJOL)

    DNA extraction is difficult in a variety of plants because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. Here we describe a modified procedure based on the hexadecyltrimethylammonium bromide (CTAB) method to ...

  8. Comparison of extraction fluids used with contaminated soils

    International Nuclear Information System (INIS)

    Erickson, D.C.; White, E.; Loehr, R.C.

    1991-01-01

    Five separate solutions were evaluated for use as leaching fluids with soils containing petroleum refining waste residues. The extraction fluids were: (a) water, (b) dilute hydrochloric acid, (c) 0.05 molar EDTA, (d) acetate buffer and (e) a dilute sulfuric/nitric acid mixture. The soils were collected from former refinery land treatment sites which had been used to treat petroleum refining wastes. The extractions were performed using a rotary tumbler (30 RPM, 18 hours) and the resulting solutions were analyzed for polynuclear aromatic hydrocarbons (PAHs) and metals. Concentrations of the PAHs in each of the five solutions were near or below the analytical quantitation limits. Metal concentrations were highest in the HCL and EDTA extracts, although only a small fraction of the total available metal present in the soils was extracted by the solutions evaluated

  9. The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing.

    Science.gov (United States)

    Kennedy, Nicholas A; Walker, Alan W; Berry, Susan H; Duncan, Sylvia H; Farquarson, Freda M; Louis, Petra; Thomson, John M; Satsangi, Jack; Flint, Harry J; Parkhill, Julian; Lees, Charlie W; Hold, Georgina L

    2014-01-01

    Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories. Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient. DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/µL respectively (p<0.0001). Hierarchical clustering of sequence data by Yue and Clayton coefficient revealed four clusters. Samples from individuals H3 and I2 clustered by patient; however, samples from patient I1 extracted with the MoBio kit clustered with samples from patient H4 rather than the other I1 samples. Linear modelling on relative abundance of common bacterial families revealed significant differences between kits; samples extracted with MoBio Powersoil showed significantly increased Bacteroidaceae, Ruminococcaceae and Porphyromonadaceae, and lower Enterobacteriaceae, Lachnospiraceae, Clostridiaceae, and Erysipelotrichaceae (p<0.05). This study demonstrates significant differences in DNA yield and bacterial DNA composition when comparing DNA extracted from the same fecal sample with different extraction kits. This highlights the importance of ensuring that samples

  10. DNA Everywhere. A Guide for Simplified Environmental Genomic DNA Extraction Suitable for Use in Remote Areas

    Energy Technology Data Exchange (ETDEWEB)

    Pecora, Gabrielle N. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Reid, Francine C. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Tom, Lauren M. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Piceno, Yvette M. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Andersen, Gary L. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2016-05-01

    Collecting field samples from remote or geographically distant areas can be a financially and logistically challenging. With participation of a local organization where the samples are originated from, gDNA samples can be extracted from the field and shipped to a research institution for further processing and analysis. The ability to set up gDNA extraction capabilities in the field can drastically reduce cost and time when running long-term microbial studies with a large sample set. The method outlined here has developed a compact and affordable method for setting up a “laboratory” and extracting and shipping gDNA samples from anywhere in the world. This white paper explains the process of setting up the “laboratory”, choosing and training individuals with no prior scientific experience how to perform gDNA extractions and safe methods for shipping extracts to any research institution. All methods have been validated by the Andersen group at Lawrence Berkeley National Laboratory using the Berkeley Lab PhyloChip.

  11. Phosphate fertilizers with varying water-solubility applied to Amazonian soils: II. Soil P extraction methods

    International Nuclear Information System (INIS)

    Muraoka, T.; Brasil, E.C.; Scivittaro, W.B.

    2002-01-01

    A pot experiment was carried out under greenhouse conditions at the Centro de Energia Nuclear na Agricultura, Piracicaba (SP, Brazil), to evaluate the phosphorus availability of different phosphate sources in five Amazonian soils. The soils utilized were: medium texture Yellow Latosol, clayey Yellow Latosol, very clayey Yellow Latosol, clayey Red-Yellow Podzolic and very clayey Red-Yellow Podzolic. Four phosphate sources were applied: triple superphosphate, ordinary Yoorin thermophosphate, coarse Yoorin termo-phosphate and North Carolina phosphate rock at P rates of 0, 40, 80 and 120 mg kg -1 soil. The dry matter yield and the amount of P taken up by cowpea and rice were correlated with the extractable P by anionic exchangeable resin, Mehlich-1, Mehlich-3 and Bray-I. The results showed that the extractable P by Mehlich-1 was higher in the soils amended with North Carolina rock phosphate. Irrespective of the phosphorus sources used, the Mehlich-3 extractant showed close correlation with plant response. The Mehlich-3 and Bray-I extractants were more sensitive to soil variations. The Mehlich-3 extractant was more suitable in predicting the P availability to plants in the different soils and phosphorus sources studied. (author)

  12. Direct Cellular Lysis/Protein Extraction Protocol for Soil Metaproteomics

    Energy Technology Data Exchange (ETDEWEB)

    Chourey, Karuna [ORNL; Jansson, Janet [Lawrence Berkeley National Laboratory (LBNL); Verberkmoes, Nathan C [ORNL; Shah, Manesh B [ORNL; Chavarria, Krystle L. [Lawrence Berkeley National Laboratory (LBNL); Tom, Lauren M [Lawrence Berkeley National Laboratory (LBNL); Brodie, Eoin L. [Lawrence Berkeley National Laboratory (LBNL); Hettich, Robert {Bob} L [ORNL

    2010-01-01

    We present a novel direct protocol for deep proteome characterization of microorganisms in soil. The method employs thermally assisted detergent-based cellular lysis (SDS) of soil samples, followed by TCA precipitation for proteome extraction/cleanup prior to liquid chromatography-mass spectrometric characterization. This approach was developed and optimized using different soils inoculated with genome-sequenced bacteria (Gram-negative Pseudomonas putida or Gram-positive Arthrobacter chlorophenolicus). Direct soil protein extraction was compared to protein extraction from cells isolated from the soil matrix prior to lysis (indirect method). Each approach resulted in identification of greater than 500 unique proteins, with a wide range in molecular mass and functional categories. To our knowledge, this SDS-TCA approach enables the deepest proteome characterizations of microbes in soil to date, without significant biases in protein size, localization, or functional category compared to pure cultures. This protocol should provide a powerful tool for ecological studies of soil microbial communities.

  13. An optimized DNA extraction protocol for benthic Didymosphenia geminata.

    Science.gov (United States)

    Uyua, Noelia Mariel; Manrique, Julieta Marina; Jones, Leandro Roberto

    2014-09-01

    Didymosphenia geminata mats display few cells in relation to extracellular material and contain polysaccharides and heavy metals that interfere with molecular studies. We describe an optimized DNA extraction protocol that help to overcome these difficulties. Our protocol outperformed five previously described DNA extraction techniques. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. A CTAB Procedure Of Total Genomic DNA Extraction For Medicinal Mushrooms

    International Nuclear Information System (INIS)

    Azhar Mohamad; Muhammad Hussaini Mohd Mustafa; Muhammad Hanif Azhari Noor; Rosnani Abdul Rashid; Hasan Hamdani Hasan Mutaat; Meswan Meskom; Mat Rasol Awang

    2014-01-01

    Medicinal mushroom is defined as mushrooms used in medicine or medical research. Isolation of intact, high-molecular-mass genomic DNA is essential for many molecular biology applications including Polymerase Chain Reaction (PCR), endonuclease restriction digestion, Southern blot analysis, and genomic library construction. The most important and prerequisite towards reliable molecular biology work is the total genomic DNA of a sample must be in good quality. Five freshly samples of medicinal mushroom were used in this work known as Auriculariapolytricha, Lentinus edode, Pleurotus sayorcaju, Sczhizopyllum commune and Ganodermalucidum. 5 mg of each sample were used to extraction the DNA, prepared in 3 replications and repeated twice. PCR based technique by using ISSR markers were used in checking the amplification ability of the total genomic extraction. A standard Doyle and Doyle protocol for genomic DNA extraction was modified in optimizing the total genomic DNA from the medicinal mushroom.The modification parameters were percentage of CTAB, incubation period and temperature. The results reveal that each sample required a certain combinations of time and period of incubation. Besides, percentage of CTAB in the buffer was found significant in giving a high yielding of extracted total genomic DNA. The extracted total genomic DNA from the medicinal mushroom yielded from 39.7 ng/ μl to 919.1 ng/ μl. The different yield among the samples found to be corresponded to polysaccharide content in the medicinal mushrooms. The objective of this works is to optimize total genomic DNA extraction of medicinal mushrooms towards a high quality intact genomic DNA for molecular activities. (author)

  15. Extraction of DNA from honey and its amplification by PCR for botanical identification

    Directory of Open Access Journals (Sweden)

    Sona Arun Jain

    2013-12-01

    Full Text Available The physiochemical and biological properties of honey are directly associated to its floral origin. Some current commonly used methods for identification of botanical origin of honey involve palynological analysis, chromatographic methods, or direct observation of the bee behavior. However, these methods can be less sensitive and time consuming. DNA-based methods have become popular due to their simplicity, quickness, and reliability. The main objective of this research is to introduce a protocol for the extraction of DNA from honey and demonstrate that the molecular analysis of the extracted DNA can be used for its botanical identification. The original CTAB-based protocol for the extraction of DNA from plants was modified and used in the DNA extraction from honey. DNA extraction was carried out from different honey samples with similar results in each replication. The extracted DNA was amplified by PCR using plant specific primers, confirming that the DNA extracted using the modified protocol is of plant origin and has good quality for analysis of PCR products and that it can be used for botanical identification of honey.

  16. Evaluation of five methods for total DNA extraction from western corn rootworm beetles.

    Directory of Open Access Journals (Sweden)

    Hong Chen

    Full Text Available BACKGROUND: DNA extraction is a routine step in many insect molecular studies. A variety of methods have been used to isolate DNA molecules from insects, and many commercial kits are available. Extraction methods need to be evaluated for their efficiency, cost, and side effects such as DNA degradation during extraction. METHODOLOGY/PRINCIPAL FINDINGS: From individual western corn rootworm beetles, Diabrotica virgifera virgifera, DNA extractions by the SDS method, CTAB method, DNAzol reagent, Puregene solutions and DNeasy column were compared in terms of DNA quantity and quality, cost of materials, and time consumed. Although all five methods resulted in acceptable DNA concentrations and absorbance ratios, the SDS and CTAB methods resulted in higher DNA yield (ng DNA vs. mg tissue at much lower cost and less degradation as revealed on agarose gels. The DNeasy kit was most time-efficient but was the costliest among the methods tested. The effects of ethanol volume, temperature and incubation time on precipitation of DNA were also investigated. The DNA samples obtained by the five methods were tested in PCR for six microsatellites located in various positions of the beetle's genome, and all samples showed successful amplifications. CONCLUSION/SIGNIFICANCE: These evaluations provide a guide for choosing methods of DNA extraction from western corn rootworm beetles based on expected DNA yield and quality, extraction time, cost, and waste control. The extraction conditions for this mid-size insect were optimized. The DNA extracted by the five methods was suitable for further molecular applications such as PCR and sequencing by synthesis.

  17. DNA Extraction and Primer Selection

    DEFF Research Database (Denmark)

    Karst, Søren Michael; Nielsen, Per Halkjær; Albertsen, Mads

    Talk regarding pitfalls in DNA extraction and 16S amplicon primer choice when performing community analysis of complex microbial communities. The talk was a part of Workshop 2 "Principles, Potential, and Limitations of Novel Molecular Methods in Water Engineering; from Amplicon Sequencing to -omics...

  18. Zinc species distribution in EDTA-extract residues of zinc-contaminated soil

    International Nuclear Information System (INIS)

    Chang, S.-H.; Wei, Y.-L.; Wang, H. Paul

    2007-01-01

    Soil sample from a site heavily contaminated with >10 wt.% zinc is sampled and extracted with aqueous solutions of ethylene diamine tetra-acetic acid (EDTA) that is a reagent frequently used to extract heavy metals in soil remediation. Three liquid/soil ratios (5/1, 20/1, and 100/1) were used in the extracting experiment. The molecular environment of the residual Zn in the EDTA-extract residues of zinc-contaminated soil is investigated with XANES technique. The results indicate that EDTA does not show considerable preference of chelating for any particular Zn species during the extraction. Zn species distribution in the sampled soil is found to resemble that in all EDTA-extract residues; Zn(OH) 2 is determined as the major zinc species (60-70%), seconded by organic zinc (21-26%) and zinc oxide (9-14%)

  19. Comparison of DNA extraction methods for detection of citrus huanglongbing in Colombia

    Directory of Open Access Journals (Sweden)

    Jorge Evelio Ángel

    2014-04-01

    Full Text Available Four DNA citrus plant tissue extraction protocols and three methods of DNA extraction from vector psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae were compared as part of the validation process and standardization for detection of huanglongbing (HLB. The comparison was done using several criterias such as integrity, purity and concentration. The best quality parameters presented in terms of extraction of DNA from plant midribs tissue of citrus, were cited by Murray and Thompson (1980 and Rodríguez et al. (2010, while for the DNA extraction from psyllid vectors of HLB, the best extraction method was suggested by Manjunath et al.(2008.

  20. Comparison of DNA extraction methods for human gut microbial community profiling.

    Science.gov (United States)

    Lim, Mi Young; Song, Eun-Ji; Kim, Sang Ho; Lee, Jangwon; Nam, Young-Do

    2018-03-01

    The human gut harbors a vast range of microbes that have significant impact on health and disease. Therefore, gut microbiome profiling holds promise for use in early diagnosis and precision medicine development. Accurate profiling of the highly complex gut microbiome requires DNA extraction methods that provide sufficient coverage of the original community as well as adequate quality and quantity. We tested nine different DNA extraction methods using three commercial kits (TianLong Stool DNA/RNA Extraction Kit (TS), QIAamp DNA Stool Mini Kit (QS), and QIAamp PowerFecal DNA Kit (QP)) with or without additional bead-beating step using manual or automated methods and compared them in terms of DNA extraction ability from human fecal sample. All methods produced DNA in sufficient concentration and quality for use in sequencing, and the samples were clustered according to the DNA extraction method. Inclusion of bead-beating step especially resulted in higher degrees of microbial diversity and had the greatest effect on gut microbiome composition. Among the samples subjected to bead-beating method, TS kit samples were more similar to QP kit samples than QS kit samples. Our results emphasize the importance of mechanical disruption step for a more comprehensive profiling of the human gut microbiome. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

  1. Direct DNA extraction method of an obligate parasitic fungus from infected plant tissue.

    Science.gov (United States)

    Liu, L; Wang, C L; Peng, W Y; Yang, J; Lan, M Q; Zhang, B; Li, J B; Zhu, Y Y; Li, C Y

    2015-12-28

    Powdery mildew and rust fungi are obligate parasites that cannot live without host organisms. They are difficult to culture in synthetic medium in the laboratory. Genomic DNA extraction is one of the basic molecular techniques used to study the genetic structure of populations. In this study, 2 different DNA extraction methods, Chelex-100 and cetyltrimethylammonium bromide (CTAB), were used to extract DNA from euonymus powdery mildew and Puccinia striiformis f. sp Tritici. Polymerase chain reaction was carried out with a race-specific-marker rDNA-internal transcribed spacer sequence. Both DNA extraction methods were compared and analyzed. The results showed that both Chelex-100 and CTAB were effective for extracting genomic DNA from infected plant tissue. However, less DNA was required for the Chelex-100 method than for the CTAB method, and the Chelex-100 method involved fewer steps, was simpler and safer, and did not require organic solvents compared to the CTAB method. DNA quality was evaluated by polymerase chain reaction, and the results showed that genomic DNA extracted using the Chelex-100 method was better than that using CTAB method, and was sufficient for studying the genetic structure of population.

  2. Digital PCR for direct quantification of viruses without DNA extraction.

    Science.gov (United States)

    Pavšič, Jernej; Žel, Jana; Milavec, Mojca

    2016-01-01

    DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration material and it has higher tolerance to inhibitors. DNA quantification without an extraction step (i.e. direct quantification) was performed here using dPCR and two different human cytomegalovirus whole-virus materials. Two dPCR platforms were used for this direct quantification of the viral DNA, and these were compared with quantification of the extracted viral DNA in terms of yield and variability. Direct quantification of both whole-virus materials present in simple matrices like cell lysate or Tris-HCl buffer provided repeatable measurements of virus concentrations that were probably in closer agreement with the actual viral load than when estimated through quantification of the extracted DNA. Direct dPCR quantification of other viruses, reference materials and clinically relevant matrices is now needed to show the full versatility of this very promising and cost-efficient development in virus quantification.

  3. Effect of soil contaminant extraction method in determining toxicity using the Microtox(reg.) assay

    International Nuclear Information System (INIS)

    Harkey, G.A.; Young, T.M.

    2000-01-01

    This project examined the influence of different extraction methods on the measured toxicity of contaminated soils collected from manufactured gas plant (MGP) sites differing in soil composition and contaminant concentration. Aged soils from a number of MGP sites were extracted using a saline solution, supercritical fluid extraction (SFE), and Soxhlet extraction. Toxicity was assessed using two forms of Microtox tests: acute aqueous tests on saline and SFE soil extracts and solid-phase tests (SPTs) on soil particles. Microtox SPTs were performed on soils before and after SFE to determine resulting toxicity reduction. Three hypotheses were tested: (1) Toxicity of soil extracts is related to contaminant concentrations of the extracts, (2) measured toxicity significantly decreases with less vigorous methods of extraction, and (3) supercritical fluid extractability correlates with measured toxicity. The EC50s for SPTs performed before and after SFE were not different for some soils but were significantly greater after extraction for other soils tested. The most significant toxicity reductions were observed for soils exhibiting the highest toxicity in both preextraction SPTs and acute aqueous tests. Acute Microtox tests performed on SFE extracts showed significantly lower EC50s than those reported from saline-based extraction procedures. Toxicity of the soils measured by Microtox SPTs was strongly correlated with both SFE efficiency and measures of contaminant aging. Data from this project provide evidence of sequestration and reduced availability of polycyclic aromatic hydrocarbons (PAHs) from soils extracted via physiologically based procedures compared to vigorous physical extraction protocols

  4. Soil and groundwater remediation using dual-phase extraction technology

    International Nuclear Information System (INIS)

    Miller, A.W.; Gan, D.R.

    1995-01-01

    A gasoline underground storage tank (UST) was formerly used to fuel vehicles for a hospital in Madison, Wisconsin. Elevated concentrations of gasoline range organics (GRO) were observed in soils and groundwater at the site during the tank removal and a subsequent site investigation. Based on the extent of soil and groundwater contamination, a dual-phase extraction technology was selected as the most cost effective alternative to remediate the site. The dual-phase extraction system includes one extraction well functioning both as a soil vapor extraction (SVE) and groundwater recovery well. After six months of operation, samples collected from the groundwater monitoring wells indicated that the groundwater has been cleaned up to levels below the Wisconsin preventative action limits. The dual-phase extraction system effectively remediated the site in a short period of time, saving both operation and maintenance costs and overall project cost

  5. [Removal of volatile organic compounds in soils by soil vapor extraction (SVE)].

    Science.gov (United States)

    Yin, Fu-xiang; Zhang, Sheng-tian; Zhao, Xin; Feng, Ke; Lin, Yu-suo

    2011-05-01

    An experiment study has been carried out to investigate effects of the diameter of soil columns, the size of soil particulate and different contaminants on efficiency of simulated soil vapor extraction (SVE). Experiments with benzene, toluene, ethylbenzene and n-propylbenzene contaminated soils showed that larger bottom area/soil height (S/H) of the columns led to higher efficiency on removal of contaminants. Experiments with contaminated soils of different particulate size showed that the efficiency of SVE decreased with increases in soil particulate size, from 10 mesh to between 20 mesh and 40 mesh and removal of contaminants in soils became more difficult. Experiments with contaminated soils under different ventilation rates suggested that soil vapor extraction at a ventilation rate of 0.10 L x min(-1) can roughly remove most contaminants from the soils. Decreasing of contaminants in soils entered tailing stages after 12 h, 18 h and 48 h for benzene, toluene and ethylbenzene, respectively. Removal rate of TVOCs (Total VOCs) reached a level as high as 99.52%. The results of the experiment have indicated that molecule structure and properties of the VOCs are also important factors which have effects on removal rates of the contaminants. Increases in carbon number on the benzene ring, decreases in vapor pressure and volatile capability resulted in higher difficulties in soil decontamination. n-propylbenzene has a lower vapor pressure than toluene and ethylbenzene which led to a significant retard effect on desorption and volatilization of benzene and ethylbenzene.

  6. Detection of Bacillus anthracis in the air, soil and animal tissue

    Directory of Open Access Journals (Sweden)

    Kušar D.

    2012-01-01

    Full Text Available The objective of the present work was to establish effective and rapid diagnostic methods for the detection of Bacillus anthracis, a highly virulent zoonotic pathogen, in the air, soil and animal (or human tissue samples. Liquid culture of B. anthracis was aerosolized and four air sampling procedures were employed. Detection of B. anthracis in the air samples was successful with RCS High Flow sampler (culturebased detection and when sampling through the air filter (molecular detection using SmartHelix Complex Samples DNA Extraction Kit. Liquid B. anthracis culture was also employed for spiking the homogenised bovine lymphatic gland tissue and soil samples. DNA extraction was performed using three different commercial kits for each sample type. High Pure PCR Template Preparation Kit was the most effective for DNA extraction from animal tissue samples. Detection in the soil was successful when PowerSoil DNA Isolation Kit was used. Our results indicate that B. anthracis can be monitored in different matrices by rapid molecular methods when appropriate sampling and DNA extraction procedures are employed prior to PCR assay. The selected rapid protocols can be implemented in specialized veterinary or human diagnostic laboratories with moderate costs.

  7. Two mini-preparation protocols to DNA extraction from plants with ...

    African Journals Online (AJOL)

    Were standardized two previously reported standard plant DNA extraction methods, but improved them on mini preparations to use the samples for population genetic analysis. The combination of CTAB lysis procedure-solvent extraction and DNA column purification (DNeasy plant mini kit modification) enables a faster and ...

  8. A rapid and low-cost DNA extraction method for isolating ...

    African Journals Online (AJOL)

    The price of commercial DNA extraction methods makes the routine use of polymerase chain reaction amplification (PCR) based methods rather costly for scientists in developing countries. A guanidium thiocayante-based DNA extraction method was investigated in this study for the isolation of Escherichia coli (E. coli) DNA ...

  9. A novel method of genomic DNA extraction for Cactaceae1

    Science.gov (United States)

    Fehlberg, Shannon D.; Allen, Jessica M.; Church, Kathleen

    2013-01-01

    • Premise of the study: Genetic studies of Cactaceae can at times be impeded by difficult sampling logistics and/or high mucilage content in tissues. Simplifying sampling and DNA isolation through the use of cactus spines has not previously been investigated. • Methods and Results: Several protocols for extracting DNA from spines were tested and modified to maximize yield, amplification, and sequencing. Sampling of and extraction from spines resulted in a simplified protocol overall and complete avoidance of mucilage as compared to typical tissue extractions. Sequences from one nuclear and three plastid regions were obtained across eight genera and 20 species of cacti using DNA extracted from spines. • Conclusions: Genomic DNA useful for amplification and sequencing can be obtained from cactus spines. The protocols described here are valuable for any cactus species, but are particularly useful for investigators interested in sampling living collections, extensive field sampling, and/or conservation genetic studies. PMID:25202521

  10. Dependence of the concentrations of "1"3"7Cs and potassium in extracted soil solutions on soil humidity before centrifugation

    International Nuclear Information System (INIS)

    Prorok, V.V.; Datsenko, O.Yi.; Bulavyin, L.A.; Zlens'kij, S.Je.; Melnichenko, L.Yu.; Rozuvan, S.G.; Poperenko, L.V.; White, P.J.

    2017-01-01

    Concentrations of 137Cs and potassium in solutions extracted by centrifugation from soils selected at some experimental sites in the 10-km Exclusion Zone of Chornobyl Nuclear Plant were determined. The results showed that for the majority of investigated soils, the concentration of 137Cs in soil solution depends on the humidity of the soil before centrifugation. It is possible to explain the dependence of the concentration of 137Cs in the soil solution on soil humidity from the dependence of the concentrations of molecules of different molecular-gravimetric fractions in soil solution on soil humidity. Considerable amount of 137Cs in soil solution is associated with these molecules, that is why the concentration of 137Cs in the extracted soil solution changes with the humidity of soil. These dependences differ between soils. For the majority of investigated soils the concentration of 137Cs in the extracted soil solution increases with increasing humidity of the soil. By contrast, soil humidity had no effect on the potassium concentration in the extracted soil solution for any soil investigated. It is concluded, that potassium is practically not associated with molecules of different molecular-gravimetric fractions in the extracted soil solutions

  11. A quick DNA extraction protocol: Without liquid nitrogen in ambient ...

    African Journals Online (AJOL)

    Marker assisted selection is an effective technique for quality traits selection in breeding program which are impossible by visual observation. Marker assisted selection in early generation requires rapid DNA extraction protocol for large number of samples in a low cost approach. A rapid and inexpensive DNA extraction ...

  12. Global Distribution of Plant-Extractable Water Capacity of Soil (Dunne)

    Data.gov (United States)

    National Aeronautics and Space Administration — Plant-extractable water capacity of soil is the amount of water that can be extracted from the soil to fulfill evapotranspiration demands. This data set provides an...

  13. DNA adduct quantification in Eisenia fetida after subchronic exposures to creosote contaminated soils

    Energy Technology Data Exchange (ETDEWEB)

    Charrois, J.W.A.; McGill, W.B. [Alberta Univ., Dept. of Renewable Resources, Edmonton, AB (Canada)

    1999-07-01

    Within soil ecosystems contaminant toxicity can vary from acute and chronic, depending on the time of exposure. Due to the long times involved chronic toxicity is difficult to determine. DNA adducts fall into the category of biochemical markers that act as an early warning system in environmental monitoring. It has been proposed that they could be used as a sensitive method to determine environmental exposures to compounds such as polycyclic aromatic hydrocarbons (PAHs), which can occur, although not exclusively, in creosote. In this connection, Benzo[a]pyrene (BaP) is a PAH that can be transformed into an electrophilic metabolite, which ultimately results in DNA adduct formation. Use was made of a 32P postlabeling method to quantify the number of DNA adducts occurring in the earthworm Eisenia fetida after exposure to weathered creosote contaminated- and biotreated-soils with and without additions of extra BaP. DNA adducts can be measured in earthworms exposed to creosote contaminated- and biotreated-soils. E. fetida exposed to weathered creosote contaminated soils had significantly more DNA adducts than those exposed to a pristine control soil. Exposures to creosote contaminated soils with additional BaP (1000 mg/kg) or biotreatment did not yield statistically significant increases in DNA adducts compared to the pristine control. (Abstract only)

  14. Evaluating the efficacy of DNA differential extraction methods for sexual assault evidence.

    Science.gov (United States)

    Klein, Sonja B; Buoncristiani, Martin R

    2017-07-01

    Analysis of sexual assault evidence, often a mixture of spermatozoa and victim epithelial cells, represents a significant portion of a forensic DNA laboratory's case load. Successful genotyping of sperm DNA from these mixed cell samples, particularly with low amounts of sperm, depends on maximizing sperm DNA recovery and minimizing non-sperm DNA carryover. For evaluating the efficacy of the differential extraction, we present a method which uses a Separation Potential Ratio (SPRED) to consider both sperm DNA recovery and non-sperm DNA removal as variables for determining separation efficiency. In addition, we describe how the ratio of male-to-female DNA in the sperm fraction may be estimated by using the SPRED of the differential extraction method in conjunction with the estimated ratio of male-to-female DNA initially present on the mixed swab. This approach may be useful for evaluating or modifying differential extraction methods, as we demonstrate by comparing experimental results obtained from the traditional differential extraction and the Erase Sperm Isolation Kit (PTC © ) procedures. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. DNA Extraction from Chorionic Villi for Prenatal Diagnosis of Foetal ...

    African Journals Online (AJOL)

    BACKGROUND: Extraction of DNA from the chorionic villi is the first major step in the molecular determination of foetal haemoglobin genotype. There are few reports on DNA extraction from the chorionic villi. A desired method should be simple to conduct, reliable and cost effective. OBJECTIVE: The aim of the study was to ...

  16. Effect of ageing on benzo[a]pyrene extractability in contrasting soils

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Luchun [CERAR-Centre for Environmental Risk Assessment and Remediation and Cooperative Research Centre for Contamination Assessment and Remediation of the Environment (CRC CARE), Building X, University of South Australia, Mawson Lakes, SA 5095 (Australia); Naidu, Ravi, E-mail: Ravi.Naidu@newcastle.edu.au [CERAR-Centre for Environmental Risk Assessment and Remediation and Cooperative Research Centre for Contamination Assessment and Remediation of the Environment (CRC CARE), Building X, University of South Australia, Mawson Lakes, SA 5095 (Australia); Liu, Yanju; Palanisami, Thavamani; Dong, Zhaomin; Mallavarapu, Megharaj [CERAR-Centre for Environmental Risk Assessment and Remediation and Cooperative Research Centre for Contamination Assessment and Remediation of the Environment (CRC CARE), Building X, University of South Australia, Mawson Lakes, SA 5095 (Australia); Semple, Kirk T. [Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ (United Kingdom)

    2015-10-15

    Highlights: • In vitro assessment of B[a]P in contaminated soils using 4 different methods. • An exponential kinetic model fits well with the extractability data. • Fitting parameter and {sup 14}C residue correlates with key soil properties. • Fractionation of B[a]P was obtained based on extractability by extractants. - Abstract: Changes in benzo[a]pyrene (B[a]P) extractability over 160 days ageing in four contrasting soils varying in organic matter content and clay mineralogy were investigated using dichloromethane: acetone 1:1 (DCM/Ace), 60 mM hydroxypropyl-β-cyclodextrin (HPCD) solution, 1-butanol (BuOH) and Milli-Q water. The B[a]P extractability by the four methods decreased with ageing and a first-order exponential model could be used to describe the kinetics of release. Correlation of the kinetic rate constant with major soil properties showed a significant effect of clay and sand contents and pore volume fraction (<6 nm) on sequestration of the desorbable fraction (by HPCD) and the water-extractable fraction. Analysis of {sup 14}C-B[a]P in soils after ageing showed a limited loss of B[a]P via degradation. Fractionation of B[a]P pools associated with the soil matrix was analysed according to extractability of B[a]P by the different extraction methods. A summary of the different fractions is proposed for the illustration of the effect of ageing on different B[a]P-bound fractions in soils. This study provides a better understanding of the B[a]P ageing process associated with different fractions and also emphasises the extraction capacity of the different methods employed.

  17. Effect of ageing on benzo[a]pyrene extractability in contrasting soils

    International Nuclear Information System (INIS)

    Duan, Luchun; Naidu, Ravi; Liu, Yanju; Palanisami, Thavamani; Dong, Zhaomin; Mallavarapu, Megharaj; Semple, Kirk T.

    2015-01-01

    Highlights: • In vitro assessment of B[a]P in contaminated soils using 4 different methods. • An exponential kinetic model fits well with the extractability data. • Fitting parameter and 14 C residue correlates with key soil properties. • Fractionation of B[a]P was obtained based on extractability by extractants. - Abstract: Changes in benzo[a]pyrene (B[a]P) extractability over 160 days ageing in four contrasting soils varying in organic matter content and clay mineralogy were investigated using dichloromethane: acetone 1:1 (DCM/Ace), 60 mM hydroxypropyl-β-cyclodextrin (HPCD) solution, 1-butanol (BuOH) and Milli-Q water. The B[a]P extractability by the four methods decreased with ageing and a first-order exponential model could be used to describe the kinetics of release. Correlation of the kinetic rate constant with major soil properties showed a significant effect of clay and sand contents and pore volume fraction (<6 nm) on sequestration of the desorbable fraction (by HPCD) and the water-extractable fraction. Analysis of 14 C-B[a]P in soils after ageing showed a limited loss of B[a]P via degradation. Fractionation of B[a]P pools associated with the soil matrix was analysed according to extractability of B[a]P by the different extraction methods. A summary of the different fractions is proposed for the illustration of the effect of ageing on different B[a]P-bound fractions in soils. This study provides a better understanding of the B[a]P ageing process associated with different fractions and also emphasises the extraction capacity of the different methods employed

  18. Extraction of Pentachlorophenol from Soils using Environmentally Benign Lactic Acid Solutions

    Science.gov (United States)

    Soil contamination with pentachlorophenol (PCP) is widespread across the globe. Soil washing/extraction is a common technique to remove this compound. Several soil washing/extraction solutions have been used but a majority of them have the problem of persistence in the environmen...

  19. Alkaline Extraction of DNA from Pathogenic Fungi for PCR-RFLP Analysis

    OpenAIRE

    Matsumoto, Masaru; Mishima, Shinobu; Matsuyama, Nobuaki; 松元, 賢; 松山, 宣明

    1997-01-01

    For the preparation of DNA samples from fungal mycelia alkaline extraction method was applied and assessed its usefulness for PCR-RFLP analysis. Using alkaline treatment protocols, 18S ribosomal DNAs (rDNA) derived from fungal genomic DNA of Pyricularia oryzae, P. zingiberi, Rhizoctonia solani and R. oryzae were PCR-amplified and digested with Hha I, Msp I and Hae ill. RFLP analysis with HhaI showed the divergent polymorphism between genus Pyricularia and Rhizoctonia. The alkaline DNA extract...

  20. Genomic DNA extraction protocols from ovine hair

    Directory of Open Access Journals (Sweden)

    Jennifer Nonato da Silva Prate

    2013-12-01

    Full Text Available Genomic DNA extracted from animal cells can be used for several purposes, for example, to know genetic variability and genetic relationships between individuals, breeds and/or species, paternity tests, to describe the genetic profile for registration of the animal at association of breeders, detect genetic polymorphisms (SNP related to characteristics of commercial interest, disease diagnose, assess resistance or susceptibility to pathogens, etc. For such evaluations, in general, DNA is amplified by PCR (polymerase chain reaction, and then subjected to various techniques as RFLP (restriction fragments length polymorphism, SSCP (single strand conformation polymorphism, and sequencing. The DNA may be obtained from blood, buccal swabs, meat, cartilage or hair bulb. Among all, the last biological material has been preferred by farmers for its ease acquisition. Several methods for extracting DNA from hair bulb were reported without any consensus for its implementation. This study aimed to optimize a protocol for efficient DNA extraction for use in PCR-RFLP analysis of the Prion gene. For this study, were collected hair samples containing hair bulb from 131 Santa Inês sheep belonging to the Institute of Zootechny, Nova Odessa - SP. Two DNA extraction protocols were evaluated. The first, called phenol-chloroform-isoamyl alcohol (PCIA has long been used by Animal Genetic Laboratories, whose procedures are described below: in each microtube (1.5 mL containing 500 µL of TE-Tween solution (Tris-HCl 50 mM, EDTA 1 mM and 0.5% Tween 20 were added to approximately 30 hair bulb per animal which was incubated at 65°C with shaking at 170 rpm for 2 hours. Then was added 15 µL of proteinase K [10 mg mL-1] and incubated at 55°C at 170 rpm for 6-12 hours. At the end of digestion was added 1 volume of solution phenol-chloroform-isoamyl alcohol (25:24:1 followed by vigorous shaking for 10 seconds and centrifuged at 8000 rpm and 4°C for 10 minutes. The upper phase

  1. Quantification of Paratrichodorus allius in DNA extracted from soil using TaqMan probe and SYBR green real-time PCR assays

    Science.gov (United States)

    The ectoparasitic stubby root nematode Paratrichodorus allius transmits Tobacco rattle virus, which causes corky ringspot disease resulting in significant economic losses in the potato industry. This study developed a diagnostic method for direct quantification of P. allius from soil DNA using a Taq...

  2. Acute toxicity assessment of explosive-contaminated soil extracting solution by luminescent bacteria assays.

    Science.gov (United States)

    Xu, Wenjie; Jiang, Zhenming; Zhao, Quanlin; Zhang, Zhenzhong; Su, Hongping; Gao, Xuewen; Ye, Zhengfang

    2016-11-01

    Explosive-contaminated soil is harmful to people's health and the local ecosystem. The acute toxicity of its extracting solution was tested by bacterial luminescence assay using three kinds of luminescent bacteria to characterize the toxicity of the soil. An orthogonal test L 16 (4 5 ) was designed to optimize the soil extracting conditions. The optimum extracting conditions were obtained when the ultrasonic extraction time, ultrasonic extraction temperature, and the extraction repeat times were 6 h, 40 °C, and three, respectively. Fourier transform infrared spectroscopy (FTIR) results showed that the main components of the contaminated soil's extracting solution were 2,4-dinitrotoluene-3-sulfonate (2,4-DNT-3-SO 3 - ); 2,4-dinitrotoluene-5-sulfonate (2,4-DNT-5-SO 3 - ); and 2,6-dinitrotoluene (2,6-DNT). Compared with Photobacterium phosphoreum and Vibrio fischeri, Vibrio qinghaiensis sp. Nov. is more suitable for assessing the soil extracting solution's acute toxicity. Soil washing can remove most of the contaminants toxic to luminescent bacterium Vibrio qinghaiensis sp. Nov., suggesting that it may be a potential effective remediation method for explosive-contaminated soil.

  3. Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants

    Directory of Open Access Journals (Sweden)

    Yang Moon-Sik

    2004-09-01

    Full Text Available Abstract Background DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants. Results We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1. After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes. Conclusion This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction.

  4. An improved protocol and a new grinding device for extraction of genomic DNA from microorganisms by a two-step extraction procedure.

    Science.gov (United States)

    Zhang, S S; Chen, D; Lu, Q

    2012-05-21

    Current protocols to extract genomic DNA from microorganisms are still laborious, tedious and costly, especially for the species with thick cell walls. In order to improve the effectiveness of extracting DNA from microbial samples, a novel protocol, defined as two-step extraction method, along with an improved tissue-grinding device, was developed. The protocol included two steps, disruption of microbial cells or spores by grinding the sample together with silica sand in a new device and extraction of DNA with an effective buffer containing cell lysis chemicals. The device was prepared by using a commercial electric mini-grinder, adapted with a grinding stone, and a sample cup processed by lathing from a polytetrafluoroethylene rod. We tested the method with vegetative cells of four microbial species and two microbial spores that have thick cell walls and are therefore hard to process; these included Escherichia coli JM109, Bacillus subtilis WB600, Sacchromyces cerevisiae INVSc1, Trichoderma viride AS3.3711, and the spores of S. cerevisiae and T. viride, respectively, representing Gram-positive bacteria, Gram-negative bacteria, yeast, filamentous fungi. We found that this new method and device extracted usable quantities of genomic DNA from the samples. The DNA fragments that were extracted exceeded 23 kb. The target sequences up to about 5 kb were successfully and exclusively amplified by PCR using extracted DNA as the template. In addition, the DNA extraction was finalized within 1.5 h. Thus, we conclude that this two-step extraction method is an effective and improved protocol for extraction of genomic DNA from microbial samples.

  5. Rare earth elements in soil extracts by ICP-MS

    Energy Technology Data Exchange (ETDEWEB)

    Tobler, L.; Furrer, V.; Wyttenbach, A. [Paul Scherrer Inst. (PSI), Villigen (Switzerland); Burger, M.; Jakob, A. [AC-Laboratorium Spiez (Switzerland)

    1997-09-01

    Three different horizons of a soil profile were extracted with water and with a complexing solution. 14 REEs were determined in the extracts. The distribution patterns obtained from the different horizons were rather similar and did not show the large fractionations observed between different plant species growing on this soil. (author) 2 figs., 1 ref.

  6. Respiration-to-DNA ratio reflects physiological state of microorganisms in root-free and rhizosphere soil

    Science.gov (United States)

    Blagodatskaya, E.; Blagodatsky, S.; Kuzyakov, Y.

    2009-04-01

    The double-stranded DNA (dsDNA) content in soil can serve as a measure of microbial biomass under near steady-state conditions and quantitatively reflect the exponential microbial growth initiated by substrate addition. The yield of respired CO2 per microbial biomass unit (expressed as DNA content) could be a valuable physiological indicator reflecting state of soil microbial community. Therefore, investigations combining both analyses of DNA content and respiration of soil microorganisms under steady-state and during periods of rapid growth are needed. We studied the relationship between CO2 evolution and microbial dsDNA content in native and glucose-amended samples of root-free and rhizosphere soil under Beta vulgaris (Cambisol, loamy sand from the field experiment of the Institute of Agroecology FAL, Braunschweig, Germany). Quantity of dsDNA was determined by direct DNA isolation from soil with mechanic and enzymatic disruption of microbial cell walls with following spectrofluorimetric detection with PicoGreen (Blagodatskaya et al., 2003). Microbial biomass and the kinetic parameters of microbial growth were estimated by dynamics of the CO2 emission from soil amended with glucose and nutrients (Blagodatsky et al., 2000). The CO2 production rate was measured hourly at 22оС using an automated infrared-gas analyzer system. The overall increase in microbial biomass, DNA content, maximal specific growth rate and therefore, in the fraction of microorganisms with r-strategy were observed in rhizosphere as compared to bulk soil. The rhizosphere effect for microbial respiration, biomass and specific growth rate was more pronounced for plots with half-rate of N fertilizer compared to full N addition. The DNA content was significantly lower in bulk compared to rhizosphere soil both before and during microbial growth initiated by glucose amendment. Addition of glucose to the soil strongly increased the amount of CO2 respired per DNA unit. Without substrate addition the

  7. Extraction of high quality DNA from seized Moroccan cannabis resin (Hashish.

    Directory of Open Access Journals (Sweden)

    Moulay Abdelaziz El Alaoui

    Full Text Available The extraction and purification of nucleic acids is the first step in most molecular biology analysis techniques. The objective of this work is to obtain highly purified nucleic acids derived from Cannabis sativa resin seizure in order to conduct a DNA typing method for the individualization of cannabis resin samples. To obtain highly purified nucleic acids from cannabis resin (Hashish free from contaminants that cause inhibition of PCR reaction, we have tested two protocols: the CTAB protocol of Wagner and a CTAB protocol described by Somma (2004 adapted for difficult matrix. We obtained high quality genomic DNA from 8 cannabis resin seizures using the adapted protocol. DNA extracted by the Wagner CTAB protocol failed to give polymerase chain reaction (PCR amplification of tetrahydrocannabinolic acid (THCA synthase coding gene. However, the extracted DNA by the second protocol permits amplification of THCA synthase coding gene using different sets of primers as assessed by PCR. We describe here for the first time the possibility of DNA extraction from (Hashish resin derived from Cannabis sativa. This allows the use of DNA molecular tests under special forensic circumstances.

  8. Sequential Application of Soil Vapor Extraction and Bioremediation Processes for the Remediation of Ethylbenzene-Contaminated Soils

    DEFF Research Database (Denmark)

    Soares, António Carlos Alves; Pinho, Maria Teresa; Albergaria, José Tomás

    2012-01-01

    Soil vapor extraction (SVE) is an efficient, well-known and widely applied soil remediation technology. However, under certain conditions it cannot achieve the defined cleanup goals, requiring further treatment, for example, through bioremediation (BR). The sequential application of these technol......Soil vapor extraction (SVE) is an efficient, well-known and widely applied soil remediation technology. However, under certain conditions it cannot achieve the defined cleanup goals, requiring further treatment, for example, through bioremediation (BR). The sequential application...

  9. Using DTPA-extractable soil fraction to assess the bioconcentration factor of plants in phytoremediation of urban soils

    Science.gov (United States)

    Rodríguez-Bocanegra, Javier; Roca, Núria; Tume, Pedro; Bech, Jaume

    2017-04-01

    Urban soils may be highly contaminated with potentially toxic metals, as a result of intensive anthropogenic activities. Developing cities are increasing the number of lands where is practiced the urban agriculture. In this way, it is necessary to assess the part of heavy metals that is transferred to plants in order to a) know the potential health risk that represent soils and b) know the relation soil-plant to assess the ability of these plants to remove heavy metals from soil. Nowadays, to assess the bioconcentration factor (BF) of plants in phytoremediation, the pseudototal o total concentration has been used by many authors. Two different urban soils with similar pH and carbonates content but with different pollution degree were phytoremediated with different plant species. Urban soil from one Barcelona district (Spain), the most contaminated soil, showed an extractability of Cu, Pb and Zn of 9.6, 6.7 and 5.8% of the total fraction respectively. The soil from Talcahuano city (Chile), with contents of heavy metals slightly above the background upper limit, present values of 15.5, 13.5 and 12% of the total fraction of studied heavy metals. Furthermore, a peri-urban analysed soil from Azul (Argentina) also showed an elevated extractability with values of 24, 13.5 and 14% of the Cu, Pb and Zn contents respectively. These soils presented more extractability than other disturbed soils, like for example, soils from mine areas. The urban soils present more developed soil with an interaction between solution and solid phase in polluted systems. The most important soil surface functional groups include the basal plane of phyllosilicates and metal hydroxyls at edge sites of clay minerals, iron oxyhydroxides, manganese oxyhydroxides and organic matter. The interaction between solution and solid phase in polluted urban systems tends to form labile associations and pollutants are more readily mobilized because their bonds with soil particles are weaker. Clay and organic

  10. Estimating the extractability of potentially toxic metals in urban soils: A comparison of several extracting solutions

    International Nuclear Information System (INIS)

    Madrid, F.; Reinoso, R.; Florido, M.C.; Diaz Barrientos, E.; Ajmone-Marsan, F.; Davidson, C.M.; Madrid, L.

    2007-01-01

    Metals released by the extraction with aqua regia, EDTA, dilute HCl and sequential extraction (SE) by the BCR protocol were studied in urban soils of Sevilla, Torino, and Glasgow. By multivariate analysis, the amounts of Cu, Pb and Zn liberated by any method were statistically associated with one another, whereas other metals were not. The mean amounts of all metals extracted by HCl and by SE were well correlated, but SE was clearly underestimated by HCl. Individual data for Cu, Pb and Zn by both methods were correlated only if each city was considered separately. Other metals gave poorer relationships. Similar conclusions were reached comparing EDTA and HCl, with much lower values for EDTA. Dilute HCl extraction cannot thus be recommended for general use as alternative to BCR SE in urban soils. - Dilute HCl extraction is tested as an alternative to the BCR sequential extraction in urban soils

  11. COMPARISON OF COMMERCIAL DNA KITS AND TRADITIONAL DNA EXTRACTION PROCEDURE IN PCR DETECTION OF PORK IN DRY/FERMENTED SAUSAGES

    Directory of Open Access Journals (Sweden)

    Ivona Djurkin Kušec

    2015-09-01

    Full Text Available In the present study four commercially available DNA extraction kits (Wizard® Genomic DNA Purification Kit, High Pure PCR Template Kit, DNeasy mericon Food and GeneJET PCR Purification Kit, as well as standard phenol/chloroform isolation technique have been evaluated regarding their concentration, purity and suitability for amplification of porcine DNA in dry/fermented sausages. The isolates were assessed for quantity and quality using spectrophotometer (IMPLEN GmbH, Germany. To verify template usability and quality of isolated DNA, the polymerase chain reaction (PCR targeting at porcine cytochrome b by species specific primers was used. The comparison of extraction methods revealed satisfactory efficiency and purity of all extraction kits, while with standard phenol/chloroform isolation method high concentrations of DNA with low A260/280 were obtained. However, all the investigated techniques proved to be suitable for identification of porcine DNA in dry/fermented sausage. Thus, the standard phenol/chloroform DNA extraction method, as the cost-effective one, can be recommended as a good alternative to more expensive isolation kits when investigating the presence of pork DNA in dry/ fermented meat products.

  12. Toxicity Thresholds Based on EDTA Extractable Nickel and Barley Root Elongation in Chinese Soils

    Directory of Open Access Journals (Sweden)

    Guangyun Zhu

    2018-04-01

    Full Text Available The uncertainty in the risk assessment of trace metal elements in soils when total metal contents are used can be decreased by assessing their availability and/or extractability when the soils have a high background value or different sources of trace metal elements. In this study, the added water-soluble nickel (Ni toxicity to barley root elongation was studied in 17 representative Chinese soil samples with and without artificial rainwater leaching. The extractability of added Ni in soils was estimated by three sequential extractions with ethylenediaminetetraacetic acid (EDTA. The results showed that the effective concentration of EDTA extractable Ni (EC50, which caused 50% inhibition of barley root elongation, ranged from 46 to 1019 mg/kg in unleached soils and 24 to 1563 mg/kg in leached soils. Regression models for EDTA extractable Ni and total Ni added to soils against soil properties indicated that EDTA extractable Ni was significantly correlated with the total Ni added to soils and that pH was the most important control factor. Regression models for toxicity thresholds based on EDTA extractable Ni against soil properties showed that soil citrate dithionate extractable Fe was more important than soil pH in predicting Ni toxicity. These results can be used to accurately assess the risk of contaminated soils with high background values and/or different Ni sources.

  13. DNA extraction and barcode identification of development stages of forensically important flies in the Czech Republic.

    Science.gov (United States)

    Olekšáková, Tereza; Žurovcová, Martina; Klimešová, Vanda; Barták, Miroslav; Šuláková, Hana

    2018-04-01

    Several methods of DNA extraction, coupled with 'DNA barcoding' species identification, were compared using specimens from early developmental stages of forensically important flies from the Calliphoridae and Sarcophagidae families. DNA was extracted at three immature stages - eggs, the first instar larvae, and empty pupal cases (puparia) - using four different extraction methods, namely, one simple 'homemade' extraction buffer protocol and three commercial kits. The extraction conditions, including the amount of proteinase K and incubation times, were optimized. The simple extraction buffer method was successful for half of the eggs and for the first instar larval samples. The DNA Lego Kit and DEP-25 DNA Extraction Kit were useful for DNA extractions from the first instar larvae samples, and the DNA Lego Kit was also successful regarding the extraction from eggs. The QIAamp DNA mini kit was the most effective; the extraction was successful with regard to all sample types - eggs, larvae, and pupari.

  14. Evaluation of a simple, non-alkaline extraction protocol to quantify soil ergosterol

    NARCIS (Netherlands)

    De Ridder-Duine, A.S.; Smant, W.; Van der Wal, A.; Van Veen, J.A.; De Boer, W.

    2006-01-01

    Quantification of soil ergosterol is increasingly used as an estimate for soil fungal biomass. Several methods for extraction of ergosterol from soil have been published, perhaps the simplest being that described by Gong, P., Guan, X., Witter, E. [2001. A rapid method to extract ergosterol from soil

  15. DNA extraction from hair shafts of wild Brazilian felids and canids.

    Science.gov (United States)

    Alberts, C C; Ribeiro-Paes, J T; Aranda-Selverio, G; Cursino-Santos, J R; Moreno-Cotulio, V R; Oliveira, A L D; Porchia, B F M M; Santos, W F; Souza, E B

    2010-12-21

    Wild felids and canids are usually the main predators in the food chains where they dwell and are almost invisible to behavior and ecology researchers. Due to their grooming behavior, they tend to swallow shed hair, which shows up in the feces. DNA found in hair shafts can be used in molecular studies that can unravel, for instance, genetic variability, reproductive mode and family structure, and in some species, it is even possible to estimate migration and dispersion rates in given populations. First, however, DNA must be extracted from hair. We extracted successfully and dependably hair shaft DNA from eight wild Brazilian felids, ocelot, margay, oncilla, Geoffroy's cat, pampas cat, jaguarundi, puma, and jaguar, as well as the domestic cat and from three wild Brazilian canids, maned wolf, crab-eating fox, and hoary fox, as well as the domestic dog. Hair samples came mostly from feces collected at the São Paulo Zoo and were also gathered from non-sedated pet or from recently dead wild animals and were also collected from museum specimens. Fractions of hair samples were stained before DNA extraction, while most samples were not. Our extraction protocol is based on a feather DNA extraction technique, based in the phenol:chloroform:isoamyl alcohol general method, with proteinase K as digestive enzyme.

  16. Availability of heavy metals in contaminated soil evidenced by chemical extractants

    Directory of Open Access Journals (Sweden)

    Maria Ligia de Souza Silva

    2012-06-01

    Full Text Available Heavy metals have been accumulating in Brazilian soils, due to natural processes, such as atmospheric deposition, or human industrial activities. For certain heavy metals, when in high concentrations in the soil, there is no specific extractant to determine the availability of these elements in the soil. The objective of the present study was to evaluate the availability of Cd, Cu, Fe, Mn, Pb and Zn for rice and soybeans, using different chemical extractants. In this study we used seven soil samples with different levels of contamination, in completely randomized experimental design with four replications. We determined the available concentrations of Cd, Cu, Fe, Mn, Pb and Zn extracted by Mehlich-1, HCl 0.1 mol L-1, DTPA, and organic acid extractants and the contents in rice and soybeans, which extracts were analyzed by ICP-OES. It was observed that Mehlich-1, HCl 0.1 mol L-1 and DTPA extractants were effective to assess the availability of Cd, Cu, Pb and Zn for rice and soybeans. However, the same was not observed for the organic acid extractant.

  17. Carcinogenicity of soil extracts

    Energy Technology Data Exchange (ETDEWEB)

    Shcherbak, N P

    1970-01-01

    A total of 270 3-mo-old mice, hybrids of the C57BL and CBA strains which are highly susceptible to carcinogens, were painted on the skin (2-3 admin./week) with 3-4 drops of (1) a concentrated benzene extract of soil taken near a petroleum refinery with a 3,4 benzpyrene (BP) content of 0.22%; (2) a 0.22% soln of pure BP in benzene; (3) a concentrated benzene extract of soil taken from an old residential area of Moscow (BP content 0.0004%); (4) a 0.0004% BP soln in benzene; and (5) pure benzene. Only mice in the first 2 groups developed tumors. In group (1), 8 mice had papillomas, 46 had skin cancer, 1 had a sarcoma and 2 had plasmocytomas. In group (2) all 60 animals had skin cancer. Lung metastases were present at autopsy in 5 mice in group (1) and in 10 mice in group (2); in some cases, these tumors were multiple. Lymph node metastases were found in 6 mice in group (1) and in 10 mice in group (2). Tumors developed more slowly in group (1) than in group (2).

  18. Detection of Ophiocordyceps sinensis in soil by quantitative real-time PCR.

    Science.gov (United States)

    Peng, Qingyun; Zhong, Xin; Lei, Wei; Zhang, Guren; Liu, Xin

    2013-03-01

    Ophiocordyceps sinensis, one of the best known entomopathogenic fungi in traditional Chinese medicine, parasitizes larvae of the moth genus Thitarodes, which lives in soil tunnels. However, little is known about the spatial distribution of O. sinensis in the soil. We established a protocol for DNA extraction, purification, and quantification of O. sinensis in soil with quantitative real-time PCR targeting the internal transcribed spacer region. The method was assessed using 34 soil samples from Tibet. No inhibitory effects in purified soil DNA extracts were detected. The standard curve method for absolute DNA quantification generated crossing point values that were strongly and linearly correlated to the log10 of the initial amount of O. sinensis genomic DNA (r(2) = 0.999) over 7 orders of magnitude (4 × 10(1) to 4 × 10(7) fg). The amplification efficiency and y-intercept value of the standard curve were 1.953 and 37.70, respectively. The amount of O. sinensis genomic DNA decreased with increasing soil depth and horizontal distance from a sclerotium (P protocol is rapid, specific, sensitive, and provides a powerful tool for quantification of O. sinensis from soil.

  19. Use of sequential extraction to assess metal partitioning in soils

    International Nuclear Information System (INIS)

    Kaasalainen, Marika; Yli-Halla, Markku

    2003-01-01

    The state of heavy metal pollution and the mobility of Cd, Cu, Ni, Cr, Pb and Zn were studied in three texturally different agricultural soil profiles near a Cu-Ni smelter in Harjavalta, Finland. The pseudo-total concentrations were determined by an aqua regia procedure. Metals were also determined after division into four fractions by sequential extraction with (1) acetic acid (exchangeable and specifically adsorbed metals), (2) a reducing agent (bound to Fe/Mn hydroxides), (3) an oxidizing agent (bound to soil organic matter) and (4) aqua regia (bound to mineral structures). Fallout from the smelter has increased the concentrations of Cd, Cu and Ni in the topsoil, where 75-90% of Cd, 49-72% of Cu and 22-52% of Ni occurred in the first two fractions. Slight Pb and Zn pollution was evident as well. High proportions of mobile Cd, Cu and Ni also deeper in the sandy soil, closest to the smelter, indicated some downward movement of metals. The hydroxide-bound fraction of Pb dominated in almost all soils and horizons, while Ni, Cr and Zn mostly occurred in mineral structures. Aqua regia extraction is usefully supplemented with sequential extraction, particularly in less polluted soils and in soils that exhibit substantial textural differences within the profiles. - Sequential extraction is most useful with soils with low metal pollutant levels

  20. Effect of DNA extraction and sample preservation method on rumen bacterial population.

    Science.gov (United States)

    Fliegerova, Katerina; Tapio, Ilma; Bonin, Aurelie; Mrazek, Jakub; Callegari, Maria Luisa; Bani, Paolo; Bayat, Alireza; Vilkki, Johanna; Kopečný, Jan; Shingfield, Kevin J; Boyer, Frederic; Coissac, Eric; Taberlet, Pierre; Wallace, R John

    2014-10-01

    The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Chemical extraction to assess the bioavailability of chlorobenzenes in soil with different aging periods

    Energy Technology Data Exchange (ETDEWEB)

    Song, Yang; Wang, Fang; Yang, Xinglun; Liu, Cuiying; Jin, Xin; Jiang, Xin [Chinese Academy of Sciences, Nanjing (China). State Key Lab. of Soil and Sustainable Agriculture; Kengara, Fredrick Orori [Maseno Univ. (Kenya). Dept. of Chemistry

    2011-12-15

    Bioavailability is mainly influenced by aging and desorption of contaminants in soil. The purpose of this study was to investigate the desorption kinetics of chlorobenzenes (CBs) in soil and to investigate whether chemical extractions are suitable for the bioavailability assessment of CBs in soil. A soil spiked with CBs and aged for different periods was extracted with Tenax, hydroxypropyl-{beta}-cyclodextrin (HPCD), and butanol to assess the bioavailability of CBs in soil, respectively. Earthworm (Eisenia foetida) accumulation was used as bioassay in parallel experiments to evaluate the chemical extractions. The results showed that desorption of CBs from soil with consecutive Tenax extraction fitted into triphasic kinetics model. Different chemical methods extracted different amounts of CBs over different aging periods. For hexachlorobenzene (HCB), the extraction efficiency was in the order of butanol > Tenax-6h > HPCD extraction, while the order of butanol > HPCD > Tenax-6h extraction for pentachlorobenzene (PeCB). The bioaccumulation by earthworm decreased with increasing aging period and was significantly higher for HCB than for PeCB (p < 0.05). Earthworm accumulated CBs correlated well with all the three chemical extracted CBs. However, HPCD extraction showed the converse extraction tendency with earthworm uptake of CBs. Chemical extraction could be used to assess the bioavailability of contaminants in soil; however, they were method and compound specific. Tenax and butanol extractions were more reliable than HPCD extraction for bioavailability assessment of the tested CBs and the soil used since they showed the consistent extraction tendency with earthworm uptake of CBs.

  2. Chemical elemental distribution and soil DNA fingerprints provide the critical evidence in murder case investigation.

    Directory of Open Access Journals (Sweden)

    Giuseppe Concheri

    Full Text Available BACKGROUND: The scientific contribution to the solution of crime cases, or throughout the consequent forensic trials, is a crucial aspect of the justice system. The possibility to extract meaningful information from trace amounts of samples, and to match and validate evidences with robust and unambiguous statistical tests, are the key points of such process. The present report is the authorized disclosure of an investigation, carried out by Attorney General appointment, on a murder case in northern Italy, which yielded the critical supporting evidence for the judicial trial. METHODOLOGY/PRINCIPAL FINDINGS: The proportional distribution of 54 chemical elements and the bacterial community DNA fingerprints were used as signature markers to prove the similarity of two soil samples. The first soil was collected on the crime scene, along a corn field, while the second was found in trace amounts on the carpet of a car impounded from the main suspect in a distant location. The matching similarity of the two soils was proven by crossing the results of two independent techniques: a elemental analysis via inductively coupled plasma mass spectrometry (ICP-MS and optical emission spectrometry (ICP-OES approaches, and b amplified ribosomal DNA restriction analysis by gel electrophoresis (ARDRA. CONCLUSIONS: Besides introducing the novel application of these methods to forensic disciplines, the highly accurate level of resolution observed, opens new possibilities also in the fields of soil typing and tracking, historical analyses, geochemical surveys and global land mapping.

  3. Comparison of two silica-based extraction methods for DNA isolation from bones.

    Science.gov (United States)

    Rothe, Jessica; Nagy, Marion

    2016-09-01

    One of the most demanding DNA extractions is from bones and teeth due to the robustness of the material and the relatively low DNA content. The greatest challenge is due to the manifold nature of the material, which is defined by various factors, including age, storage, environmental conditions, and contamination with inhibitors. However, most published protocols do not distinguish between different types or qualities of bone material, but are described as being generally applicable. Our laboratory works with two different extraction methods based on silica membranes or the use of silica beads. We compared the amplification success of the two methods from bone samples with different qualities and in the presence of inhibitors. We found that the DNA extraction using the silica membrane method results an in higher DNA yield but also in a higher risk of co-extracting impurities, which can act as inhibitors. In contrast the silica beads method shows decreased co-extraction of inhibitors but also less DNA yield. Related to our own experiences it has to be considered that each bone material should be reviewed independently regarding the analysis and extraction method. Therefore, the most ambitious task is determining the quality of the bone material, which requires substantial experience. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. Human DNA Extraction by Two Extraction Methods for Forensic Typification from Human Feces on FTA Paper

    Directory of Open Access Journals (Sweden)

    Shirleny Monserrat Sandoval-Arias

    2014-11-01

    Full Text Available The identification of suspects in criminal investigations has been facilitated since DNA test are executed on different samples. The application of this technology for forensic typification from human fecal samples still presents complications therefore this research evaluated two DNA extraction protocols with modifications to determine that of major efficiency. Organic extractions and extractions using the commercial kit “IQTM DNA Casework Sample Kit for Maxwell ® 16” on FTA portions of 4cm2 and 1cm2 impregnated with feces from the same individual were done to accomplish the objective. In all the assays the results were useful, however; the best forensic typification (by the electropherogram characteristics was obtained by using the commercial kit in an area of 1 cm2 of FTA paper impregnated in a 1:4 dilution.

  5. Arduino-based automation of a DNA extraction system.

    Science.gov (United States)

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile.

  6. Effect of Cassia hirsuta (L) extract on DNA profile of some ...

    African Journals Online (AJOL)

    The effect of ethanol extract of leaf of Cassia hirsute (L) on the DNA profile of some selected pathogenic microorganisms were investigated using PCR-RAPD analysis to generate DNA fingerprint. The change in molecular configuration of organisms with and without extract shows a wide disparity between the sensitive and ...

  7. Comparison of different methodologies for DNA extraction from Aegla longirostri

    Directory of Open Access Journals (Sweden)

    João Vitor Trindade Bitencourt

    2007-11-01

    Full Text Available The aim of this study was to compare some DNA extraction methodologies for Aegla longirostri. The protocols were based on the traditional phenol-chloroform DNA extraction methodology and using a commercial kit for DNA extraction. They differed in tissues used, the addition - or not - of beta-mercaptoethanol to the lysis buffer, times and methods for the animal's conservation (frozen, in ethanol or fresh. Individuals stored at -20°C for a long time supplied lower molecular weight DNA than those stored for a short time. The best yield for the specimens preserved in ethanol was obtained for 15 days storage in 95% ethanol. The kit resulted in a low quantity of high molecular weight DNA. The best protocol for DNA extraction from Aeglidae, and probably for other crustaceans should, therefore, utilize fresh specimens, with addition of beta-mercaptoethanol to the lysis buffer.Marcadores moleculares são ferramentas úteis para esclarecer dúvidas a respeito dos Aeglidae, único grupo de crustáceos Anomura de água doce. Essas técnicas dependem da obtenção de DNA de boa qualidade e sem contaminantes. O objetivo deste estudo foi comparar algumas metodologias de extração de DNA de Aegla longirostri. Quatorze protocolos foram analisados, baseados na metodologia tradicional de extração de DNA com fenol-clorofórmio, exceto o protocolo K no qual se utilizou um Kit. Os procedimentos diferiram quanto aos tecidos utilizados e a adição de beta-mercaptoetanol ao tampão de lise. Avaliaram-se também diferentes tempos e maneiras de conservação. Indivíduos congelados apresentaram maior degradação do material obtido conforme o tempo em que ficaram congelados. Para os indivíduos conservados em álcool, aqueles mantidos em etanol 95% forneceram material de melhor qualidade. A utilização do Mini Kit resultou em uma quantidade muito pequena de DNA de alto peso molecular. O melhor protocolo para extração de DNA de Aeglidae utilizou músculos e br

  8. Element fractionation by sequential extraction in a soil with high carbonate content

    International Nuclear Information System (INIS)

    Sulkowski, Margareta; Hirner, Alfred V.

    2006-01-01

    The influence of carbonate and other buffering substances in soils on the results of a 3-step sequential extraction procedure (BCR) used for metal fractionation was investigated. Deviating from the original extraction scheme, where the extracts are analysed only for a limited number of metals, almost all elements in the soils were quantified by X-ray fluorescence spectroscopy, in the initial samples as well as in the residues of all extraction steps. Additionally, the mineral contents were determined by X-ray diffractometry. Using this methodology, it was possible to correlate changes in soil composition caused by the extraction procedure with the release of elements. Furthermore, the pH values of all extracts were monitored, and certain extraction steps were repeated until no significant pH-rise occurred. A soil with high dolomite content (27%) and a carbonate free soil were extracted. Applying the original BCR-sequence to the calcareous soil, carbonate was found in the residues of the first two steps and extract pH-values rose by around two units in the first and second step, caused mainly by carbonate dissolution. This led to wrong assignment of the carbonate elements Ca, Mg, Sr, Ba, and also to decreased desorption and increased re-adsorption of ions in those steps. After repetition of the acetic acid step until extract pH remained low, the carbonate was completely destroyed and the distributions of the elements Ca, Mg, Sr, Ba as well as those of Co, Ni, Cu, Zn and Pb were found to be quite different to those determined in the original extraction. Furthermore, it could be shown that the effectiveness of the reduction process in step two was reduced by increasing pH: Fe oxides were not significantly attacked by the repeated acetic acid treatments, but a 10-fold amount of Fe was mobilized by hydroxylamine hydrochloride after complete carbonate destruction. On the other hand, only small amounts of Fe were released anyway. Even repeated reduction steps did not

  9. Quantitative Field Testing Rotylenchulus reniformis DNA from Metagenomic Samples Isolated Directly from Soil

    Science.gov (United States)

    Showmaker, Kurt; Lawrence, Gary W.; Lu, Shien; Balbalian, Clarissa; Klink, Vincent P.

    2011-01-01

    A quantitative PCR procedure targeting the β-tubulin gene determined the number of Rotylenchulus reniformis Linford & Oliveira 1940 in metagenomic DNA samples isolated from soil. Of note, this outcome was in the presence of other soil-dwelling plant parasitic nematodes including its sister genus Helicotylenchus Steiner, 1945. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from soil. PMID:22194958

  10. Remediation of uranium contaminated soils with bicarbonate extraction and microbial U(VI) reduction

    Science.gov (United States)

    Philips , Elizabeth J.P.; Landa, Edward R.; Lovely, Derek R.

    1995-01-01

    A process for concentrating uranium from contaminated soils in which the uranium is first extracted with bicarbonate and then the extracted uranium is precipitated with U(VI)-reducing microorganisms was evaluated for a variety of uranuum-contaminated soils. Bicarbonate (100 mM) extracted 20–94% of the uranium that was extracted with nitric acid. The U(VI)-reducing microorganism,Desulfovibrio desulfuricans reduced the U(VI) to U(IV) in the bicarbonate extracts. In some instances unidentified dissolved extracted components, presumably organics, gave the extract a yellow color and inhibited U(VI) reduction and/or the precipitation of U(IV). Removal of the dissolved yellow material with the addition of hydrogen peroxide alleviated this inhibition. These results demonstrate that bicarbonate extraction of uranium from soil followed by microbial U(VI) reduction might be an effective mechanism for concentrating uranium from some contaminated soils.

  11. Remediation of uranium contaminated soils with bicarbonate extraction and microbial U(VI) reduction

    International Nuclear Information System (INIS)

    Phillips, E.J.P.; Landa, E.R.; Lovley, D.R.

    1995-01-01

    A process for concentrating uranium from contaminated soils in which the uranium is first extracted with bicarbonate and then the extracted uranium is precipitated with U(VI)-reducing microorganisms was evaluated for a variety of uranium-contaminated soils. Bicarbonate (100 mM) extracted 20-94% of the uranium that was extracted with nitric acid. The U(VI)-reducing microorganism, Desulfovibrio desulfuricans reduced the U(VI) to U(IV) in the bicarbonate extracts. In some instances unidentified dissolved extracted components, presumably organics, gave the extract a yellow color and inhibited U(VI) reduction and/or the precipitation of U(IV). Removal of the dissolved yellow material with the addition of hydrogen peroxide alleviated this inhibition. These results demonstrate that bicarbonate extraction of uranium from soil followed by microbial U(VI) reduction might be an effective mechanism for concentrating uranium from some contaminated soils. (author)

  12. Combined in-situ and ex-situ bioremediation of petroleum hydrocarbon contaminated soils by closed-loop soil vapor extraction and air injection

    International Nuclear Information System (INIS)

    Hu, S.S.; Buckler, M.J.

    1993-01-01

    Treatment and restoration of petroleum hydrocarbon contaminated soils at a bulk petroleum above-ground storage tank (AST) site in Michigan is being conducted through in-situ and ex-situ closed-loop soil vapor extraction (SVE), soil vapor treatment, and treated air injection (AI) processes. The soil vapor extraction process applies a vacuum through the petroleum hydrocarbon affected soils in the ex-situ bio-remediation pile (bio-pile) and along the perimeter of excavated area (in-situ area) to remove the volatile or light petroleum hydrocarbons. This process also draws ambient air into the ex-situ bio-pile and in-situ vadose zone soil along the perimeter of excavated area to enhance biodegradation of light and heavy petroleum hydrocarbons in the soil. The extracted soil vapor is treated using a custom-designed air bio-remediation filter (bio-filter) to degrade the petroleum hydrocarbon compounds in the soil vapor extraction air streams. The treated air is then injected into a flush grade soil bed in the backfill area to perform final polishing of the air stream, and to form a closed-loop air flow with the soil vapor extraction perforated pipes along the perimeter of the excavated area

  13. Sequential extraction for the speciation of some heavy metals in soils

    International Nuclear Information System (INIS)

    Zemberyova, M.; Zwaik, A.A.H.; Farkasovska, I.

    1998-01-01

    The five step sequential extraction for speciation of copper and nickel originally designed for sediments has been applied to soil samples. The extractant solutions were: 1 mol/l ammonium acetate, 1 mol/l hydroxylammonium chloride in 25% acetic acid (1:1), 0.1 mol/l hydrochlorid acid, 0.5 mol/l sodium hydroxide and 8 mol/l nitric acid. The residue was decomposed by HF and HNO 3 . Using this procedure the metal fraction bound to the organic matter can be distinguished. The concentrations of analytes were determined in the soil extracts by FAAS and ETAAS. Accuracy was assessed by comparing the sum of the contents of copper and nickel in soil extracts with the total certified values of CRMs of soils. The overall recovery values for nickel was 84-105% and for copper 105-114%. (author)

  14. Comparison of protocols for genomic DNA extraction from 'velame ...

    African Journals Online (AJOL)

    usuario

    2013-07-24

    Jul 24, 2013 ... involving C. linearifolius, we compared the efficiency of six protocols for genomic DNA extraction previously ... phytic, with diverse aspect and floristics, average rainfall between ..... The variation observed for DNA concentrations estimated with .... performed with protocol 1 (data not shown), or still, bands.

  15. Modifying and adapting a plant-based DNA extraction protocol for ...

    African Journals Online (AJOL)

    ... a 100 apparently healthy individuals residing in Calabar. The modified DNA procedure yielded good quality genomic DNA which was used in carrying out allele specific polymerase chain reaction which also yielded good quality amplicons. This method is simple and suitable for the extraction of DNA from human red cell.

  16. DNA extraction from wings as a suitable approach for queen bees genotyping

    Directory of Open Access Journals (Sweden)

    Elena Facchini

    2018-06-01

    Full Text Available In livestock, genomics has been used since a decade in combination with phenotypic information for the estimation of breeding values. In honey bees (Apis mellifera, the advantage for including genomics in selective breeding programmes is represented by the possibility to reduce the generation interval and increase the accuracies of estimated breeding values resulting in higher genetic gain (Brascamp et al., 2018. The limit for this application is DNA extraction. Extraction methods for small animals such as insects often rely upon destructive approaches. The challenge is to develop tissue sampling methods that permit the survival of the animal while providing adequate quality DNA for genotyping. Along with previous reports of DNA extraction from several matrices, this study aims to contribute in developing suitable methodologies for genotyping honey bees queens using DNA extracted from wing cuttings (Chaline et al., 2004; Gregory and Rinderer, 2004; Gould et al., 2011. The clipping of the queen wings in beekeeping is a common practice and it ensures the survival and normal activities of the animal (Forster, 1971. A total of 57 queens with known pedigree were enrolled for this study. Wings from each queen were cut and stored at -20°C until processed (Fig. 1. Extractions were carried out using a modified protocol provided by Qiagen (DNeasy® Blood & Tissue. The modification consists in an initial incubation of the samples with proteinase K for 20 minutes, further steps are carried out following the manufacturer’s instructions. To test the suitability of the extracted DNA for genotyping, PCR was performed on Esterase FE4 like gene. Although quantification with NanoDrop™ resulted in <20 ng/μL of DNA in solution, the extracted material was sufficient for PCR amplification of candidate genes for sequencing and genotyping. Our results show that it is possible to extract DNA from wings’ cuttings permitting to implement genomic approaches in honey

  17. Genomic DNA extraction from sapwood of Pinus roxburghii for ...

    African Journals Online (AJOL)

    Ashish

    2013-02-22

    Feb 22, 2013 ... A method for extraction of genomic DNA from sapwood tissues of mature tall trees of Pinus roxburghii, .... DNA as a template. PCR was performed on a thermal cycler. (Biorad, Mycycler) incorporating 10 ng genomic DNA to a 25 µl reaction mix containing 1X Taq buffer, 3 mM MgCl2, 0.2 mM each of dNTPs ...

  18. Effect of DNA extraction in the Rosa canina L. identification under different processing temperature

    Directory of Open Access Journals (Sweden)

    Jana Žiarovská

    2017-01-01

    Full Text Available Rosa canina, L. is widely used for medicinal purposes as well as in food industry where it is a valuable source, bioactive compounds and natural colorants. Actually, no specific method is recommended as suitable one for DNA extraction from rose hips. The aim of the study was to compare three commercial and three non-commercial methods to extract total genomic DNA from rose hips hyphanthium. Four methods are based on the precipitation in principle and two methods are based on resin-binding. Extracted DNA was proved for the effectivity in following PCR. In total, six different DNA isolations was performed for differently heat processes rose hips - fresh hyphanthium, 2-weeks frozen hyphanthium, dried hyphanthium (50 °C and boiled hyphanthium (100 °C. The amplification parameters of 500 bp chloroplast gene amplicon were evaluated. Obtained amounts of extracted DNA was very variable not only for every individual method used but for individual treatment of samples, too. In general, non-commercial method provided higher amount of extracted DNA, but the A260/280 ratio was lower. When regarding the processing treatment of the samples, high differences were found among the samples untreated by heat and those that were dried or boiled for three of the used extraction methods. All the samples were positive for amplification, but different amounts of amplified product were obtained. The comparison of data for concentrations of extracted DNA and concentrations of amplified product showed large differences when regarding the achieved purity of DNA in extraction.

  19. Bioremediation of petroleum hydrocarbon contaminated soils using soil vapor extraction: Case study

    International Nuclear Information System (INIS)

    Roth, R.J.; Peterson, R.M.

    1994-01-01

    Soils contaminated with petroleum hydrocarbons are being remediated in situ at a site in Lakewood, New Jersey by bioremediation in conjunction with soil vapor extractions (SVE) and nutrient addition. The contaminants were from hydraulic oils which leaked from subsurface hydraulic lifts, waste oil from leaking underground storage tanks (USTs), an aboveground storage tank, and motor oil from a leaking UST. The oils contaminated subsurface soils at the site to a depth of 25 feet. Approximately 900 cubic yards of soil were contaminated. Soil sample analyses showed total petroleum hydrocarbon (TPH) concentrations up to 31,500 ppm. The design of the remedial system utilized the results of a treatability study which showed that TPH degrading microorganisms, when supplied with oxygen and nutrients, affected a 14% reduction in TPH in 30 days. A SVE system was installed which used three wells, each installed to a depth of 25 feet below grade. The SVE system was operated to achieve an extracted air flow of approximately 20 to 30 scfm from each well. Bioremediation of the TPH was monitored by measuring CO 2 and O 2 concentrations at the wellheads and vapor monitoring probes. After four months of remediation, CO 2 concentrations were at a minimum, at which point the subsurface soils were sampled and analyzed for TPH. The soil analyses showed a removal of TPH by biodegradation of up to 99.8% after four months of remediation

  20. Profiling nematode communities in unmanaged flowerbed and agricultural field soils in Japan by DNA barcode sequencing.

    Directory of Open Access Journals (Sweden)

    Hisashi Morise

    Full Text Available Soil nematodes play crucial roles in the soil food web and are a suitable indicator for assessing soil environments and ecosystems. Previous nematode community analyses based on nematode morphology classification have been shown to be useful for assessing various soil environments. Here we have conducted DNA barcode analysis for soil nematode community analyses in Japanese soils. We isolated nematodes from two different environmental soils of an unmanaged flowerbed and an agricultural field using the improved flotation-sieving method. Small subunit (SSU rDNA fragments were directly amplified from each of 68 (flowerbed samples and 48 (field samples isolated nematodes to determine the nucleotide sequence. Sixteen and thirteen operational taxonomic units (OTUs were obtained by multiple sequence alignment from the flowerbed and agricultural field nematodes, respectively. All 29 SSU rDNA-derived OTUs (rOTUs were further mapped onto a phylogenetic tree with 107 known nematode species. Interestingly, the two nematode communities examined were clearly distinct from each other in terms of trophic groups: Animal predators and plant feeders were markedly abundant in the flowerbed soils, in contrast, bacterial feeders were dominantly observed in the agricultural field soils. The data from the flowerbed nematodes suggests a possible food web among two different trophic nematode groups and plants (weeds in the closed soil environment. Finally, DNA sequences derived from the mitochondrial cytochrome oxidase c subunit 1 (COI gene were determined as a DNA barcode from 43 agricultural field soil nematodes. These nematodes were assigned to 13 rDNA-derived OTUs, but in the COI gene analysis were assigned to 23 COI gene-derived OTUs (cOTUs, indicating that COI gene-based barcoding may provide higher taxonomic resolution than conventional SSU rDNA-barcoding in soil nematode community analysis.

  1. Profiling Nematode Communities in Unmanaged Flowerbed and Agricultural Field Soils in Japan by DNA Barcode Sequencing

    Science.gov (United States)

    Morise, Hisashi; Miyazaki, Erika; Yoshimitsu, Shoko; Eki, Toshihiko

    2012-01-01

    Soil nematodes play crucial roles in the soil food web and are a suitable indicator for assessing soil environments and ecosystems. Previous nematode community analyses based on nematode morphology classification have been shown to be useful for assessing various soil environments. Here we have conducted DNA barcode analysis for soil nematode community analyses in Japanese soils. We isolated nematodes from two different environmental soils of an unmanaged flowerbed and an agricultural field using the improved flotation-sieving method. Small subunit (SSU) rDNA fragments were directly amplified from each of 68 (flowerbed samples) and 48 (field samples) isolated nematodes to determine the nucleotide sequence. Sixteen and thirteen operational taxonomic units (OTUs) were obtained by multiple sequence alignment from the flowerbed and agricultural field nematodes, respectively. All 29 SSU rDNA-derived OTUs (rOTUs) were further mapped onto a phylogenetic tree with 107 known nematode species. Interestingly, the two nematode communities examined were clearly distinct from each other in terms of trophic groups: Animal predators and plant feeders were markedly abundant in the flowerbed soils, in contrast, bacterial feeders were dominantly observed in the agricultural field soils. The data from the flowerbed nematodes suggests a possible food web among two different trophic nematode groups and plants (weeds) in the closed soil environment. Finally, DNA sequences derived from the mitochondrial cytochrome oxidase c subunit 1 (COI) gene were determined as a DNA barcode from 43 agricultural field soil nematodes. These nematodes were assigned to 13 rDNA-derived OTUs, but in the COI gene analysis were assigned to 23 COI gene-derived OTUs (cOTUs), indicating that COI gene-based barcoding may provide higher taxonomic resolution than conventional SSU rDNA-barcoding in soil nematode community analysis. PMID:23284767

  2. Rapid and efficient method to extract metagenomic DNA from estuarine sediments.

    Science.gov (United States)

    Shamim, Kashif; Sharma, Jaya; Dubey, Santosh Kumar

    2017-07-01

    Metagenomic DNA from sediments of selective estuaries of Goa, India was extracted using a simple, fast, efficient and environment friendly method. The recovery of pure metagenomic DNA from our method was significantly high as compared to other well-known methods since the concentration of recovered metagenomic DNA ranged from 1185.1 to 4579.7 µg/g of sediment. The purity of metagenomic DNA was also considerably high as the ratio of absorbance at 260 and 280 nm ranged from 1.88 to 1.94. Therefore, the recovered metagenomic DNA was directly used to perform various molecular biology experiments viz. restriction digestion, PCR amplification, cloning and metagenomic library construction. This clearly proved that our protocol for metagenomic DNA extraction using silica gel efficiently removed the contaminants and prevented shearing of the metagenomic DNA. Thus, this modified method can be used to recover pure metagenomic DNA from various estuarine sediments in a rapid, efficient and eco-friendly manner.

  3. Remediation of soils combining soil vapor extraction and bioremediation: benzene.

    Science.gov (United States)

    Soares, António Alves; Albergaria, José Tomás; Domingues, Valentina Fernandes; Alvim-Ferraz, Maria da Conceição M; Delerue-Matos, Cristina

    2010-08-01

    This work reports the study of the combination of soil vapor extraction (SVE) with bioremediation (BR) to remediate soils contaminated with benzene. Soils contaminated with benzene with different water and natural organic matter contents were studied. The main goals were: (i) evaluate the performance of SVE regarding the remediation time and the process efficiency; (ii) study the combination of both technologies in order to identify the best option capable to achieve the legal clean up goals; and (iii) evaluate the influence of soil water content (SWC) and natural organic matter (NOM) on SVE and BR. The remediation experiments performed in soils contaminated with benzene allowed concluding that: (i) SVE presented (a) efficiencies above 92% for sandy soils and above 78% for humic soils; (b) and remediation times from 2 to 45 h, depending on the soil; (ii) BR showed to be an efficient technology to complement SVE; (iii) (a) SWC showed minimum impact on SVE when high airflow rates were used and led to higher remediation times for lower flow rates; (b) NOM as source of microorganisms and nutrients enhanced BR but hindered the SVE due the limitation on the mass transfer of benzene from the soil to the gas phase. (c) 2010 Elsevier Ltd. All rights reserved.

  4. DNA purification using dynamic solid-phase extraction on a rotationally-driven polyethylene-terephthalate microdevice

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, K.R. [Department of Chemistry, University of Virginia, Charlottesville, VA 22904 (United States); Borba, J.C. [Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos-SP (Brazil); Meija, M. [Department of Mechanical and Aerospace Engineering, University of Virginia, Charlottesville, VA 22904 (United States); Mills, D.L. [TeGrex Technologies, Sperryville, VA 22740 (United States); Haverstick, D.M. [Department of Pathology, University of Virginia, Charlottesville, VA 22904 (United States); Olson, K.E.; Aranda, R. [Office of the Chief Scientist, Defense Forensic Science Center, N 31st Street, Atlanta, GA 30297 (United States); Garner, G.T. [Department of Mechanical and Aerospace Engineering, University of Virginia, Charlottesville, VA 22904 (United States); Carrilho, E. [Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos-SP (Brazil); Landers, J.P., E-mail: landers@virginia.edu [Department of Chemistry, University of Virginia, Charlottesville, VA 22904 (United States); Department of Mechanical and Aerospace Engineering, University of Virginia, Charlottesville, VA 22904 (United States); Department of Pathology, University of Virginia, Charlottesville, VA 22904 (United States)

    2016-09-21

    We report the development of a disposable polyester toner centrifugal device for semi-automated, dynamic solid phase DNA extraction (dSPE) from whole blood samples. The integration of a novel adhesive and hydrophobic valving with a simple and low cost microfabrication method allowed for sequential addition of reagents without the need for external equipment for fluid flow control. The spin-dSPE method yielded an average extraction efficiency of ∼45% from 0.6 μL of whole blood. The device performed single sample extractions or accommodate up to four samples for simultaneous DNA extraction, with PCR-readiness DNA confirmed by effective amplification of a β-globin gene. The purity of the DNA was challenged by a multiplex amplification with 16 targeted amplification sites. Successful multiplexed amplification could routinely be obtained using the purified DNA collected post an on-chip extraction, with the results comparable to those obtained with commercial DNA extraction methods. This proof-of-principle work represents a significant step towards a fully-automated low cost DNA extraction device. - Highlights: • dSPE design on centrifugal PeT device with a unique mixing strategy was proposed. • Increased fluidic control with novel adhesive tape valves on a PeT device. • Multiplexed spin-dSPE device to run up to 4 samples simultaneously. • Demonstrated strong singleplexed and multiplexed amplification following chip dSPE.

  5. Meta-barcoding of 'dirt' DNA from soil reflects vertebrate biodiversity

    DEFF Research Database (Denmark)

    Andersen, Kenneth; Bird, Karen Lise; Rasmussen, Morten

    2012-01-01

    DNA molecules originating from animals and plants can be retrieved directly from sediments and have been used for reconstructing both contemporary and past ecosystems. However, the extent to which such 'dirt' DNA reflects taxonomic richness and structural diversity remains contentious. Here, we...... couple second generation high-throughput sequencing with 16S mitochondrial DNA (mtDNA) meta-barcoding, to explore the accuracy and sensitivity of 'dirt' DNA as an indicator of vertebrate diversity, from soil sampled at safari parks, zoological gardens and farms with known species compositions. PCR...

  6. Quantification of Plasmodiophora brassicae Using a DNA-Based Soil Test Facilitates Sustainable Oilseed Rape Production.

    Science.gov (United States)

    Wallenhammar, Ann-Charlotte; Gunnarson, Albin; Hansson, Fredrik; Jonsson, Anders

    2016-04-22

    Outbreaks of clubroot disease caused by the soil-borne obligate parasite Plasmodiophora brassicae are common in oilseed rape (OSR) in Sweden. A DNA-based soil testing service that identifies fields where P. brassicae poses a significant risk of clubroot infection is now commercially available. It was applied here in field surveys to monitor the prevalence of P. brassicae DNA in field soils intended for winter OSR production and winter OSR field experiments. In 2013 in Scania, prior to planting, P. brassicae DNA was detected in 60% of 45 fields on 10 of 18 farms. In 2014, P. brassicae DNA was detected in 44% of 59 fields in 14 of 36 farms, in the main winter OSR producing region in southern Sweden. P. brassicae was present indicative of a risk for >10% yield loss with susceptible cultivars (>1300 DNA copies g soil(-1)) in 47% and 44% of fields in 2013 and 2014 respectively. Furthermore, P. brassicae DNA was indicative of sites at risk of complete crop failure if susceptible cultivars were grown (>50 000 copies g(-1) soil) in 14% and 8% of fields in 2013 and 2014, respectively. A survey of all fields at Lanna research station in western Sweden showed that P. brassicae was spread throughout the farm, as only three of the fields (20%) showed infection levels below the detection limit for P.brassicae DNA, while the level was >50,000 DNA copies g(-1) soil in 20% of the fields. Soil-borne spread is of critical importance and soil scraped off footwear showed levels of up to 682 million spores g(-1) soil. Soil testing is an important tool for determining the presence of P. brassicae and providing an indication of potential yield loss, e.g., in advisory work on planning for a sustainable OSR crop rotation. This soil test is gaining acceptance as a tool that increases the likelihood of success in precision agriculture and in applied research conducted in commercial oilseed fields and at research stations. The present application highlights the importance of prevention of

  7. A rapid and low-cost DNA extraction method for isolating ...

    African Journals Online (AJOL)

    user

    2011-02-21

    Feb 21, 2011 ... Available online at http://www.academicjournals.org/AJB ... α-casein, produces PCR ready DNA at a fraction of the cost of commercial DNA extraction kits. Key words: DNA .... This experiment was performed to evaluate the efficiency of the ..... Zoetendal EG, Ben-Amor K, Akkermans AD, Abee T, De Vos WM.

  8. Comparative Study of Seven Commercial Kits for Human DNA Extraction from Urine Samples Suitable for DNA Biomarker-Based Public Health Studies

    Science.gov (United States)

    El Bali, Latifa; Diman, Aurélie; Bernard, Alfred; Roosens, Nancy H. C.; De Keersmaecker, Sigrid C. J.

    2014-01-01

    Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at −20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies. PMID:25365790

  9. Comparative study of seven commercial kits for human DNA extraction from urine samples suitable for DNA biomarker-based public health studies.

    Science.gov (United States)

    El Bali, Latifa; Diman, Aurélie; Bernard, Alfred; Roosens, Nancy H C; De Keersmaecker, Sigrid C J

    2014-12-01

    Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at -20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies.

  10. Variation of the rare earth element concentrations in the soil, soil extract and in individual plants from the same site

    International Nuclear Information System (INIS)

    Wyttenbach, A.; Tobler, L.; Furrer, V.; Schleppi, P.

    1998-01-01

    Samples of various types (spruce needles, blackberry leaves, soils, and soil extracts) have each been taken at 6 places from the same site. In addition, 4 whirls each from 2 spruce trees were sampled. Rare earth elements (REEs) were determined in these samples by neutron activation analysis with a chemical group separation. Variations between places were found to be small with soils and soil extracts, but large with plants. Variations between whirls were small. Plants neither reflected the soil nor the soil extract. Both plant species were dissimilar, but the logarithm of their ratio was a linear function of the atomic number of the REE. A negative Ce anomaly (with respect to soil) was found in both plant species. (author)

  11. Bacterial community analysis of activated sludge: an evaluation of four commonly used DNA extraction methods

    NARCIS (Netherlands)

    Vanysacker, L.; Declerck, S.A.J.; Hellemans, B.; De Meester, L.; Vankelecom, I.; Declerck, P.

    2010-01-01

    The effectiveness of three commercially available direct DNA isolation kits (Mobio, Fast, Qiagen) and one published direct DNA extraction protocol (Bead) for extracting bacterial DNA from different types of activated sludge was investigated and mutually compared. The DNA quantity and purity were

  12. Phytotoxicity of water-soluble substances from alfalfa and barley soil extracts on four crop species.

    Science.gov (United States)

    Read, J J; Jensen, E H

    1989-02-01

    Problems associated with continuously planting alfalfa (Medicago saliva L.) or seeding to thicken depleted alfalfa stands may be due to autotoxicity, an intraspecific form of allelopathy. A bioassay approach was utilized to characterize the specificity and chemical nature of phytotoxins in extracts of alfalfa soils as compared to fallow soil or soil where a cereal was the previous crop. In germination chamber experiments, water-soluble substances present in methanol extracts of soil cropped to alfalfa or barley (Hordeum vulgare L.) decreased seedling root length of alfalfa L-720, winter wheat (Triticum aestivum L. Nugaines) and radish (Raphanus sativa L. Crimson Giant). Five days after germination, seedling dry weights of alfalfa and radish in alfalfa soil extracts were lower compared to wheat or red clover (Trifolium pralense L. Kenland). Growth of red clover was not significantly reduced by soil extracts from cropped soil. Extracts of crop residue screened from soil cropped to alfalfa or barley significantly reduced seedling root length; extracts of alfalfa residue caused a greater inhibition of seedling dry weight than extracts of barely residue. A phytotoxic, unidentified substance present in extracts of crop residue screened from alfalfa soil, which inhibited seedling root length of alfalfa, was isolated by thin-layer chromatography (TLC). Residues from a soil cropped continuously to alfalfa for 10 years had the greatest phytotoxic activity.

  13. Quantification of Plasmodiophora brassicae Using a DNA-Based Soil Test Facilitates Sustainable Oilseed Rape Production

    Directory of Open Access Journals (Sweden)

    Ann-Charlotte Wallenhammar

    2016-04-01

    Full Text Available Outbreaks of clubroot disease caused by the soil-borne obligate parasite Plasmodiophora brassicae are common in oilseed rape (OSR in Sweden. A DNA-based soil testing service that identifies fields where P. brassicae poses a significant risk of clubroot infection is now commercially available. It was applied here in field surveys to monitor the prevalence of P. brassicae DNA in field soils intended for winter OSR production and winter OSR field experiments. In 2013 in Scania, prior to planting, P. brassicae DNA was detected in 60% of 45 fields on 10 of 18 farms. In 2014, P. brassicae DNA was detected in 44% of 59 fields in 14 of 36 farms, in the main winter OSR producing region in southern Sweden. P. brassicae was present indicative of a risk for >10% yield loss with susceptible cultivars (>1300 DNA copies g soil−1 in 47% and 44% of fields in 2013 and 2014 respectively. Furthermore, P. brassicae DNA was indicative of sites at risk of complete crop failure if susceptible cultivars were grown (>50 000 copies g−1 soil in 14% and 8% of fields in 2013 and 2014, respectively. A survey of all fields at Lanna research station in western Sweden showed that P. brassicae was spread throughout the farm, as only three of the fields (20% showed infection levels below the detection limit for P.brassicae DNA, while the level was >50,000 DNA copies g−1 soil in 20% of the fields. Soil-borne spread is of critical importance and soil scraped off footwear showed levels of up to 682 million spores g−1 soil. Soil testing is an important tool for determining the presence of P. brassicae and providing an indication of potential yield loss, e.g., in advisory work on planning for a sustainable OSR crop rotation. This soil test is gaining acceptance as a tool that increases the likelihood of success in precision agriculture and in applied research conducted in commercial oilseed fields and at research stations. The present application highlights the importance of

  14. Impact of two different commercial DNA extraction methods on BK virus viral load

    Directory of Open Access Journals (Sweden)

    Massimiliano Bergallo

    2016-03-01

    Full Text Available Background and aim: BK virus, a member of human polyomavirus family, is a worldwide distributed virus characterized by a seroprevalence rate of 70-90% in adult population. Monitoring of viral replication is made by evaluation of BK DNA by quantitative polymerase chain reaction. Many different methods can be applied for extraction of nucleic acid from several specimens. The aim of this study was to assess the impact of two different DNA extraction procedure on BK viral load. Materials and methods: DNA extraction procedure including the Nuclisens easyMAG platform (bioMerieux, Marcy l’Etoile, France and manual QIAGEN extraction (QIAGEN Hilden, Germany. BK DNA quantification was performed by Real Time TaqMan PCR using a commercial kit. Result and discussion: The samples capacity, cost and time spent were compared for both systems. In conclusion our results demonstrate that automated nucleic acid extraction method using Nuclisense easyMAG was superior to manual protocol (QIAGEN Blood Mini kit, for the extraction of BK virus from serum and urine specimens.

  15. Effects of seasonal and well construction variables on soil vapor extraction pilot tests

    International Nuclear Information System (INIS)

    Campbell, R.; Hudon, N.; Bass, D.

    1995-01-01

    The selection and design of an effective soil vapor extraction system is dependent upon data generated from pilot testing. Therefore, it is critical to understand factors that may affect the testing prior to selecting or designing a system. In Sebago Lake Village, Maine, two adjacent gasoline stations experienced a release. Gasoline migrated through fine sand into the groundwater and discharged to a small stream. Soil vapor extraction was investigated as a remedial alternative to reduce volatile organic compounds in the unsaturated soil. Three soil vapor extraction pilot tests were performed at one of the sites and one test at the other site. The results of the testing varied. Data collected during a summer test indicated soil vapor extraction was less likely to work. The wells tested were installed using an excavator. An adequate surface seal was not present in any of the tested wells. An additional test was performed in the winter using wells installed by a drill rig. Winter test results indicated that soil vapor extraction could be effective. Another test was performed after a horizontal soil vapor extraction system with a surface seal was installed. The results of this testing indicated that soil vapor extraction was more effective than predicted by the earlier tests. Tests performed on the other property indicated that the horizontal wells were more effective than the vertical wells. Testing results were affected by the well installation method, well construction, proximity to manmade structures, and the season in which testing was performed. Understanding factors that affect the testing is critical in selecting and designing the system

  16. Evaluation of DNA Extraction Methods Suitable for PCR-based Detection and Genotyping of Clostridium botulinum

    DEFF Research Database (Denmark)

    Auricchio, Bruna; Anniballi, Fabrizio; Fiore, Alfonsina

    2013-01-01

    in terms of cost, time, labor, and supplies. Eleven botulinum toxin–producing clostridia strains and 25 samples (10 food, 13 clinical, and 2 environmental samples) naturally contaminated with botulinum toxin–producing clostridia were used to compare 4 DNA extraction procedures: Chelex® 100 matrix, Phenol......Sufficient quality and quantity of extracted DNA is critical to detecting and performing genotyping of Clostridium botulinum by means of PCR-based methods. An ideal extraction method has to optimize DNA yield, minimize DNA degradation, allow multiple samples to be extracted, and be efficient...

  17. Distributions and concentrations of thallium in Korean soils determined by single and sequential extraction procedures.

    Science.gov (United States)

    Lee, Jin-Ho; Kim, Dong-Jin; Ahn, Byung-Koo

    2015-06-01

    The objectives of this study were to investigate the distribution of thallium in soils collected near suspected areas such as cement plants, active and closed mines, and smelters and to examine the extraction of thallium in the soils using 19 single chemical and sequential chemical extraction procedures. Thallium concentrations in soils near cement plants were distributed between 1.20 and 12.91 mg kg(-1). However, soils near mines and smelters contained relatively low thallium concentrations ranging from 0.18 to 1.09 mg kg(-1). Thallium extractability with 19 single chemical extractants from selected soils near cement plants ranged from 0.10% to 8.20% of the total thallium concentration. In particular, 1.0 M NH4Cl, 1.0 M (NH4)2SO4, and 1.0 M CH3COONH4 extracted more thallium than other extractants. Sequential fractionation results of thallium from different soils such as industrially and artificially contaminated soils varied with the soil properties, especially soil pH and the duration of thallium contamination.

  18. Electrokinetic extraction of chromate from unsaturated soils

    International Nuclear Information System (INIS)

    Mattson, E.D.; Lindgren, E.R.

    1993-01-01

    Heavy-metal contamination of soil and groundwater is a widespread problem in industrial nations. Remediation by excavation of such sites may not be cost effective or politically acceptable. Electrokinetic remediation is one possible remediation technique for in situ removal of such contaminants from unsaturated soils. Previous papers discussing the work performed by researchers at Sandia National Laboratories (SNL) and Sat-Unsat, Inc. (SUI) (Lindgren et al., 1991, 1992, 1993) focused on the transport of contaminants and dyes by electrokinetics in unsaturated soils. These experiments were conducted with graphite electrodes with no extraction system. As the contaminants migrated through the soil, they increased in concentration at the electrode creating a diffusion flux in the opposite direction. This paper discusses a technique to remove the contaminants from unsaturated soils once they have reached an electrode

  19. Electrokinetic extraction of chromate from unsaturated soils

    Energy Technology Data Exchange (ETDEWEB)

    Mattson, E.D. [SAT-UNSAT, Inc., Albuquerque, NM (United States); Lindgren, E.R. [Sandia National Labs., Albuquerque, NM (United States)

    1993-11-01

    Heavy-metal contamination of soil and groundwater is a widespread problem in industrial nations. Remediation by excavation of such sites may not be cost effective or politically acceptable. Electrokinetic remediation is one possible remediation technique for in situ removal of such contaminants from unsaturated soils. Previous papers discussing the work performed by researchers at Sandia National Laboratories (SNL) and Sat-Unsat, Inc. (SUI) (Lindgren et al., 1991, 1992, 1993) focused on the transport of contaminants and dyes by electrokinetics in unsaturated soils. These experiments were conducted with graphite electrodes with no extraction system. As the contaminants migrated through the soil, they increased in concentration at the electrode creating a diffusion flux in the opposite direction. This paper discusses a technique to remove the contaminants from unsaturated soils once they have reached an electrode.

  20. The use of carrier RNA to enhance DNA extraction from microfluidic-based silica monoliths.

    Science.gov (United States)

    Shaw, Kirsty J; Thain, Lauren; Docker, Peter T; Dyer, Charlotte E; Greenman, John; Greenway, Gillian M; Haswell, Stephen J

    2009-10-12

    DNA extraction was carried out on silica-based monoliths within a microfluidic device. Solid-phase DNA extraction methodology was applied in which the DNA binds to silica in the presence of a chaotropic salt, such as guanidine hydrochloride, and is eluted in a low ionic strength solution, such as water. The addition of poly-A carrier RNA to the chaotropic salt solution resulted in a marked increase in the effective amount of DNA that could be recovered (25ng) compared to the absence of RNA (5ng) using the silica-based monolith. These findings confirm that techniques utilising nucleic acid carrier molecules can enhance DNA extraction methodologies in microfluidic applications.

  1. Assessment of soil stabilization by chemical extraction and bioaccumulation using earthworm, Eisenia fetida

    Science.gov (United States)

    Lee, Byung-Tae; Abd Aziz, Azilah; Han, Heop Jo; Kim, Kyoung-Woong

    2014-05-01

    Soil stabilization does not remove heavy metals from contaminated soil, but lowers their exposures to ecosystem. Thus, it should be evaluated by measuring the fractions of heavy metals which are mobile and/or bioavailable in soils. The study compared several chemical extractions which intended to quantify the mobile or bioaccessible fractions with uptake and bioaccumulation by earthworm, Eisenia fetida. Soil samples were taken from the abandoned mine area contaminated with As, Cd, Cu, Pb and/or Zn. To stabilize heavy metals, the soils were amended with limestone and steel slag at 5% and 2% (w/w), respectively. All chemical extractions and earthworm tests were applied to both the contaminated and the stabilized soils with triplicates. The chemical extractions consisted of six single extractions which were 0.01M CaCl2 (unbufferred), EDTA or DTPA (chelating), TCLP (acidic), Mehlich 3 (mixture), and aqua regia (peudo-total). Sequential extractions were also applied to fractionate heavy metals in soils. In earthworm tests, worms were exposed to the soils for uptake of heavy metals. After 28 days of exposure to soils, worms were transferred to clean soils for elimination. During the tests, three worms were randomly collected at proper sampling events. Worms were rinsed with DI water and placed on moist filter paper for 48 h for depuration. Filter paper was renewed at 24 h to prevent coprophagy. The worms were killed with liquid nitrogen, dried in the oven, and digested with aqua regia for ICP-MS analysis. In addition to the bioaccumulation, several toxicity endpoints were observed such as burrowing time, mortality, cocoon production, and body weight changes. Toxicokinetics was applied to determine the uptake and elimination heavy metals by the earthworms. Bioaccumulation factor (BAF) was estimated using total metal concentrations and body burdens. Pearson correlation and simple linear regression were applied to evaluate the relationship between metal fractions by single

  2. An Optimized DNA Analysis Workflow for the Sampling, Extraction, and Concentration of DNA obtained from Archived Latent Fingerprints.

    Science.gov (United States)

    Solomon, April D; Hytinen, Madison E; McClain, Aryn M; Miller, Marilyn T; Dawson Cruz, Tracey

    2018-01-01

    DNA profiles have been obtained from fingerprints, but there is limited knowledge regarding DNA analysis from archived latent fingerprints-touch DNA "sandwiched" between adhesive and paper. Thus, this study sought to comparatively analyze a variety of collection and analytical methods in an effort to seek an optimized workflow for this specific sample type. Untreated and treated archived latent fingerprints were utilized to compare different biological sampling techniques, swab diluents, DNA extraction systems, DNA concentration practices, and post-amplification purification methods. Archived latent fingerprints disassembled and sampled via direct cutting, followed by DNA extracted using the QIAamp® DNA Investigator Kit, and concentration with Centri-Sep™ columns increased the odds of obtaining an STR profile. Using the recommended DNA workflow, 9 of the 10 samples provided STR profiles, which included 7-100% of the expected STR alleles and two full profiles. Thus, with carefully selected procedures, archived latent fingerprints can be a viable DNA source for criminal investigations including cold/postconviction cases. © 2017 American Academy of Forensic Sciences.

  3. Zinc fractionation in contaminated soils by sequential and single extractions: influence of soil properties and zinc content.

    Science.gov (United States)

    Voegelin, Andreas; Tokpa, Gerome; Jacquat, Olivier; Barmettler, Kurt; Kretzschmar, Ruben

    2008-01-01

    We studied the fractionation of zinc (Zn) in 49 contaminated soils as influenced by Zn content and soil properties using a seven-step sequential extraction procedure (F1: NH4NO3; F2: NH4-acetate, pH 6; F3: NH3OHCl, pH 6; F4: NH4-EDTA, pH 4.6; F5: NH4-oxalate, pH 3; F6: NH4-oxalate/ascorbic acid, pH 3; F7: residual). The soils had developed from different geologic materials and covered a wide range in soil pH (4.0-7.3), organic C content (9.3-102 g kg(-1)), and clay content (38-451 g kg(-1)). Input of aqueous Zn with runoff water from electricity towers during 26 to 74 yr resulted in total soil Zn contents of 3.8 to 460 mmol kg(-1). In acidic soils (n = 24; pH soils (n = 25; pH > or =6.0), most Zn was extracted in the mobilizable fraction (F2) and the intermediate fractions (F4 and F5). The extractability of Zn increased with increasing Zn contamination of the soils. The sum of mobile (F1) and mobilizable (F2) Zn was independent of soil pH, the ratio of Zn in F1 over F1+F2 plotted against soil pH, exhibited the typical shape of a pH sorption edge and markedly increased from pH 6 to pH 5, reflecting the increasing lability of mobilizable Zn with decreasing soil pH. In conclusion, the extractability of Zn from soils contaminated with aqueous Zn after decades of aging under field conditions systematically varied with soil pH and Zn content. The same trends are expected to apply to aqueous Zn released from decomposing Zn-bearing contaminants, such as sewage sludge or smelter slag. The systematic trends in Zn fractionation with varying soil pH and Zn content indicate the paramount effect of these two factors on molecular scale Zn speciation. Further research is required to characterize the link between the fractionation and speciation of Zn and to determine how Zn loading and soil physicochemical properties affect Zn speciation in soils.

  4. Sources of pre-analytical variations in yield of DNA extracted from blood samples: analysis of 50,000 DNA samples in EPIC.

    Directory of Open Access Journals (Sweden)

    Elodie Caboux

    Full Text Available The European Prospective Investigation into Cancer and nutrition (EPIC is a long-term, multi-centric prospective study in Europe investigating the relationships between cancer and nutrition. This study has served as a basis for a number of Genome-Wide Association Studies (GWAS and other types of genetic analyses. Over a period of 5 years, 52,256 EPIC DNA samples have been extracted using an automated DNA extraction platform. Here we have evaluated the pre-analytical factors affecting DNA yield, including anthropometric, epidemiological and technical factors such as center of subject recruitment, age, gender, body-mass index, disease case or control status, tobacco consumption, number of aliquots of buffy coat used for DNA extraction, extraction machine or procedure, DNA quantification method, degree of haemolysis and variations in the timing of sample processing. We show that the largest significant variations in DNA yield were observed with degree of haemolysis and with center of subject recruitment. Age, gender, body-mass index, cancer case or control status and tobacco consumption also significantly impacted DNA yield. Feedback from laboratories which have analyzed DNA with different SNP genotyping technologies demonstrate that the vast majority of samples (approximately 88% performed adequately in different types of assays. To our knowledge this study is the largest to date to evaluate the sources of pre-analytical variations in DNA extracted from peripheral leucocytes. The results provide a strong evidence-based rationale for standardized recommendations on blood collection and processing protocols for large-scale genetic studies.

  5. Comparison of methods of DNA extraction for real-time PCR in a model of pleural tuberculosis.

    Science.gov (United States)

    Santos, Ana; Cremades, Rosa; Rodríguez, Juan Carlos; García-Pachón, Eduardo; Ruiz, Montserrat; Royo, Gloria

    2010-01-01

    Molecular methods have been reported to have different sensitivities in the diagnosis of pleural tuberculosis and this may in part be caused by the use of different methods of DNA extraction. Our study compares nine DNA extraction systems in an experimental model of pleural tuberculosis. An inoculum of Mycobacterium tuberculosis was added to 23 pleural liquid samples with different characteristics. DNA was subsequently extracted using nine different methods (seven manual and two automatic) for analysis with real-time PCR. Only two methods were able to detect the presence of M. tuberculosis DNA in all the samples: extraction using columns (Qiagen) and automated extraction with the TNAI system (Roche). The automatic method is more expensive, but requires less time. Almost all the false negatives were because of the difficulty involved in extracting M. tuberculosis DNA, as in general, all the methods studied are capable of eliminating inhibitory substances that block the amplification reaction. The method of M. tuberculosis DNA extraction used affects the results of the diagnosis of pleural tuberculosis by molecular methods. DNA extraction systems that have been shown to be effective in pleural liquid should be used.

  6. Genomic DNA extraction method from Annona senegalensis Pers ...

    African Journals Online (AJOL)

    aghomotsegin

    2014-02-05

    SDS) or cetyl-methyl ammonium bromide (CTAB). The released DNA should be protected from endogenous nuclease. Ethylenediaminetetra acetate. (EDTA) is often included in the extraction buffer to chelate magnesium ions ...

  7. Chemical composition and Zn bioavailability of the soil solution extracted from Zn amended variable charge soils.

    Science.gov (United States)

    Zampella, Mariavittoria; Adamo, Paola

    2010-01-01

    A study on variable charge soils (volcanic Italian and podzolic Scottish soils) was performed to investigate the influence of soil properties on the chemical composition of soil solution. Zinc speciation, bioavailability and toxicity in the soil solution were examined. The soils were spiked with increasing amounts of Zn (0, 100, 200, 400 and 1000 mg/kg) and the soil solutions were extracted using rhizon soil moisture samplers. The pH, total organic carbon (TOC), base cations, anions, total Zn and free Zn2+ in soil solution were analysed. A rapid bioassay with the luminescent bacterium Escherichia coli HB101 pUCD607 was performed to assess Zn toxicity. The influence of soil type and Zn treatments on the chemical composition of soil solution and on Zn toxicity was considered and discussed. Different trends of total and free Zn concentrations, base cations desorption and luminescence of E. coli HB101 pUCD607 were observed. The soil solution extracted from the volcanic soils had very low total and free Zn concentrations and showed specific Zn2+/Ca2+ exchange. The soil solution from the podzolic soil had much higher total and free Zn concentrations and showed no evidence of specific Zn2+/Ca2+ exchange. In comparison with the subalkaline volcanic soils, the acidic podzol showed enhanced levels of toxic free Zn2+ and consequently stronger effects on E. coli viability.

  8. Assessment of DNA extracted from FTA® cards for use on the Illumina iSelect BeadChip

    Science.gov (United States)

    McClure, Matthew C; McKay, Stephanie D; Schnabel, Robert D; Taylor, Jeremy F

    2009-01-01

    Background As FTA® cards provide an ideal medium for the field collection of DNA we sought to assess the quality of genomic DNA extracted from this source for use on the Illumina BovineSNP50 iSelect BeadChip which requires unbound, relatively intact (fragment sizes ≥ 2 kb), and high-quality DNA. Bovine blood and nasal swab samples collected on FTA cards were extracted using the commercially available GenSolve kit with a minor modification. The call rate and concordance of genotypes from each sample were compared to those obtained from whole blood samples extracted by standard PCI extraction. Findings An ANOVA analysis indicated no significant difference (P > 0.72) in BovineSNP50 genotype call rate between DNA extracted from FTA cards by the GenSolve kit or extracted from whole blood by PCI. Two sample t-tests demonstrated that the DNA extracted from the FTA cards produced genotype call and concordance rates that were not different to those produced by assaying DNA samples extracted by PCI from whole blood. Conclusion We conclude that DNA extracted from FTA cards by the GenSolve kit is of sufficiently high quality to produce results comparable to those obtained from DNA extracted from whole blood when assayed by the Illumina iSelect technology. Additionally, we validate the use of nasal swabs as an alternative to venous blood or buccal samples from animal subjects for reliably producing high quality genotypes on this platform. PMID:19531223

  9. Assessment of DNA extracted from FTA cards for use on the Illumina iSelect BeadChip.

    Science.gov (United States)

    McClure, Matthew C; McKay, Stephanie D; Schnabel, Robert D; Taylor, Jeremy F

    2009-06-16

    As FTA cards provide an ideal medium for the field collection of DNA we sought to assess the quality of genomic DNA extracted from this source for use on the Illumina BovineSNP50 iSelect BeadChip which requires unbound, relatively intact (fragment sizes >or= 2 kb), and high-quality DNA. Bovine blood and nasal swab samples collected on FTA cards were extracted using the commercially available GenSolve kit with a minor modification. The call rate and concordance of genotypes from each sample were compared to those obtained from whole blood samples extracted by standard PCI extraction. An ANOVA analysis indicated no significant difference (P > 0.72) in BovineSNP50 genotype call rate between DNA extracted from FTA cards by the GenSolve kit or extracted from whole blood by PCI. Two sample t-tests demonstrated that the DNA extracted from the FTA cards produced genotype call and concordance rates that were not different to those produced by assaying DNA samples extracted by PCI from whole blood. We conclude that DNA extracted from FTA cards by the GenSolve kit is of sufficiently high quality to produce results comparable to those obtained from DNA extracted from whole blood when assayed by the Illumina iSelect technology. Additionally, we validate the use of nasal swabs as an alternative to venous blood or buccal samples from animal subjects for reliably producing high quality genotypes on this platform.

  10. Assessment of DNA extracted from FTA® cards for use on the Illumina iSelect BeadChip

    Directory of Open Access Journals (Sweden)

    Schnabel Robert D

    2009-06-01

    Full Text Available Abstract Background As FTA® cards provide an ideal medium for the field collection of DNA we sought to assess the quality of genomic DNA extracted from this source for use on the Illumina BovineSNP50 iSelect BeadChip which requires unbound, relatively intact (fragment sizes ≥ 2 kb, and high-quality DNA. Bovine blood and nasal swab samples collected on FTA cards were extracted using the commercially available GenSolve kit with a minor modification. The call rate and concordance of genotypes from each sample were compared to those obtained from whole blood samples extracted by standard PCI extraction. Findings An ANOVA analysis indicated no significant difference (P > 0.72 in BovineSNP50 genotype call rate between DNA extracted from FTA cards by the GenSolve kit or extracted from whole blood by PCI. Two sample t-tests demonstrated that the DNA extracted from the FTA cards produced genotype call and concordance rates that were not different to those produced by assaying DNA samples extracted by PCI from whole blood. Conclusion We conclude that DNA extracted from FTA cards by the GenSolve kit is of sufficiently high quality to produce results comparable to those obtained from DNA extracted from whole blood when assayed by the Illumina iSelect technology. Additionally, we validate the use of nasal swabs as an alternative to venous blood or buccal samples from animal subjects for reliably producing high quality genotypes on this platform.

  11. A Rapid and Cost-Effective Method for DNA Extraction from Archival Herbarium Specimens.

    Science.gov (United States)

    Krinitsina, A A; Sizova, T V; Zaika, M A; Speranskaya, A S; Sukhorukov, A P

    2015-11-01

    Here we report a rapid and cost-effective method for the extraction of total DNA from herbarium specimens up to 50-90-year-old. The method takes about 2 h, uses AMPure XP magnetic beads diluted by PEG-8000- containing buffer, and does not require use of traditional volatile components like chloroform, phenol, and liquid nitrogen. It yields up to 4 µg of total nucleic acid with high purity from about 30 mg of dry material. The quality of the extracted DNA was tested by PCR amplification of 5S rRNA and rbcL genes (nuclear and chloroplast DNA markers) and compared against the traditional chloroform/isoamyl alcohol method. Our results demonstrate that the use of the magnetic beads is crucial for extraction of DNA suitable for subsequent PCR from herbarium samples due to the decreasing inhibitor concentrations, reducing short fragments of degraded DNA, and increasing median DNA fragment sizes.

  12. Effect of seven Indian plant extracts on Fenton reaction-mediated damage to DNA constituents.

    Science.gov (United States)

    Kar, Indrani; Chattopadhyaya, Rajagopal

    2017-11-01

    The influences of substoichiometric amounts of seven plant extracts in the Fenton reaction-mediated damage to deoxynucleosides, deoxynucleoside monophosphates, deoxynucleoside triphosphates, and supercoiled plasmid DNA were studied to rationalize anticancer properties reported in some of these extracts. Extracts from Acacia catechu, Emblica officinalis, Spondias dulcis, Terminalia belerica, Terminalia chebula, as well as gallic acid, epicatechin, chebulagic acid and chebulinic acid enhance the extent of damage in Fenton reactions with all monomeric substrates but protect supercoiled plasmid DNA, compared to standard Fenton reactions. The damage to pyrimidine nucleosides/nucleotides is enhanced by these extracts and compounds to a greater extent than for purine ones in a concentration dependent manner. Dolichos biflorus and Hemidesmus indicus extracts generally do not show this enhancement for the monomeric substrates though they protect plasmid DNA. Compared to standard Fenton reactions for deoxynucleosides with ethanol, the presence of these five plant extracts render ethanol scavenging less effective as the radical is generated in the vicinity of the target. Since substoichiometric amounts of these extracts and the four compounds produce this effect, a catalytic mechanism involving the presence of a ternary complex of the nucleoside/nucleotide substrate, a plant compound and the hydroxyl radical is proposed. Such a mechanism cannot operate for plasmid DNA as the planar rings in the extract compounds cannot stack with the duplex DNA bases. These plant extracts, by enhancing Fenton reaction-mediated damage to deoxynucleoside triphosphates, slow down DNA replication in rapidly dividing cancer cells, thus contributing to their anticancer properties.

  13. Study of microtip-based extraction and purification of DNA from human samples for portable devices

    Science.gov (United States)

    Fotouhi, Gareth

    DNA sample preparation is essential for genetic analysis. However, rapid and easy-to-use methods are a major challenge to obtaining genetic information. Furthermore, DNA sample preparation technology must follow the growing need for point-of-care (POC) diagnostics. The current use of centrifuges, large robots, and laboratory-intensive protocols has to be minimized to meet the global challenge of limited access healthcare by bringing the lab to patients through POC devices. To address these challenges, a novel extraction method of genomic DNA from human samples is presented by using heat-cured polyethyleneimine-coated microtips generating a high electric field. The microtip extraction method is based on recent work using an electric field and capillary action integrated into an automated device. The main challenges to the method are: (1) to obtain a stable microtip surface for the controlled capture and release of DNA and (2) to improve the recovery of DNA from samples with a high concentration of inhibitors, such as human samples. The present study addresses these challenges by investigating the heat curing of polyethyleneimine (PEI) coated on the surface of the microtip. Heat-cured PEI-coated microtips are shown to control the capture and release of DNA. Protocols are developed for the extraction and purification of DNA from human samples. Heat-cured PEI-coated microtip methods of DNA sample preparation are used to extract genomic DNA from human samples. It is discovered through experiment that heat curing of a PEI layer on a gold-coated surface below 150°C could inhibit the signal of polymerase chain reaction (PCR). Below 150°C, the PEI layer is not completely cured and dissolved off the gold-coated surface. Dissolved PEI binds with DNA to inhibit PCR. Heat curing of a PEI layer above 150°C on a gold-coated surface prevents inhibition to PCR and gel electrophoresis. In comparison to gold-coated microtips, the 225°C-cured PEI-coated microtips improve the

  14. Assessing genetic polymorphisms using DNA extracted from cells present in saliva samples

    Directory of Open Access Journals (Sweden)

    Nemoda Zsofia

    2011-12-01

    Full Text Available Abstract Background Technical advances following the Human Genome Project revealed that high-quality and -quantity DNA may be obtained from whole saliva samples. However, usability of previously collected samples and the effects of environmental conditions on the samples during collection have not been assessed in detail. In five studies we document the effects of sample volume, handling and storage conditions, type of collection device, and oral sampling location, on quantity, quality, and genetic assessment of DNA extracted from cells present in saliva. Methods Saliva samples were collected from ten adults in each study. Saliva volumes from .10-1.0 ml, different saliva collection devices, sampling locations in the mouth, room temperature storage, and multiple freeze-thaw cycles were tested. One representative single nucleotide polymorphism (SNP in the catechol-0-methyltransferase gene (COMT rs4680 and one representative variable number of tandem repeats (VNTR in the serotonin transporter gene (5-HTTLPR: serotonin transporter linked polymorphic region were selected for genetic analyses. Results The smallest tested whole saliva volume of .10 ml yielded, on average, 1.43 ± .77 μg DNA and gave accurate genotype calls in both genetic analyses. The usage of collection devices reduced the amount of DNA extracted from the saliva filtrates compared to the whole saliva sample, as 54-92% of the DNA was retained on the device. An "adhered cell" extraction enabled recovery of this DNA and provided good quality and quantity DNA. The DNA from both the saliva filtrates and the adhered cell recovery provided accurate genotype calls. The effects of storage at room temperature (up to 5 days, repeated freeze-thaw cycles (up to 6 cycles, and oral sampling location on DNA extraction and on genetic analysis from saliva were negligible. Conclusions Whole saliva samples with volumes of at least .10 ml were sufficient to extract good quality and quantity DNA. Using

  15. Enzymatic vegetable organic extracts as soil biochemical biostimulants and atrazine extenders.

    Science.gov (United States)

    García-Martínez, Ana María; Tejada, Manuel; Díaz, Ana Isabel; Rodríguez-Morgado, Bruno; Bautista, Juan; Parrado, Juan

    2010-09-08

    The purpose of this study was to gather information on the potential effects of organic biostimulants on soil activity and atrazine biodegradation. Carob germ enzymatic extract (CGEE) and wheat condensed distiller solubles enzymatic extract (WCDS-EE) have been obtained using an enzymatic process; their main organic components are soluble carbohydrates and proteins in the form of peptides and free amino acids. Their application to soil results in high biostimulation, rapidly increased dehydrogenase, phosphatase and glucosidase activities, and an observed atrazine extender capacity due to inhibition of its mineralization. The extender capacity of both extracts is proportional to the protein/carbohydrate ratio content. As a result, these enzymatic extracts are highly microbially available, leading to two independent phenomena, fertility and an atrazine persistence that is linked to increased soil activity.

  16. Soil clean up by vapour extraction: parametrical study; Depollution des sols par extraction sous pression reduite: etude de quelques parametres

    Energy Technology Data Exchange (ETDEWEB)

    Dutheil, C.

    2003-05-15

    Soil vapour extraction is a treatment process for soils polluted by volatile organic compounds. Its principle relies on the circulation of gaseous flow in soil by the application of a depression of some hundreds milli-bars. A parametrical study has been led on a soil artificially polluted by tri-chloro-ethene. It shows that the gaseous flow rate has a slight influence on pollutants extraction yield. This is due to rate limited mass transfer processes. Soil moisture plays a negative role on treatment efficiency because of the reduction of the porosity available for the gas circulation. Tests have been performed on a soil polluted by a complex mixture of organic pollutants to elaborate a methodology of technical feasibility assessment. This methodology aims at identifying and limiting risks of site rehabilitation failure. Tests results show that soil vapour extraction was inadequate to treat the soil tested in this study because of the strong affinity between a dense organic phase (grease) and chlorinated solvents. (author)

  17. Change of the Extractability of Cadmium Added to Different Soils: Aging Effect and Modeling

    Directory of Open Access Journals (Sweden)

    Xi Zhang

    2018-03-01

    Full Text Available Ethylenediaminetetraacetic acid (EDTA is known to be a chelating agent and has been widely used for estimating the total extractable metals in soil. The effect of aging on EDTA-extractable cadmium (Cd was investigated in five different soils at three Cd concentrations incubated for 180 days. The EDTA-extractable Cd rapidly decreased after incubated during 30–60 days, followed by slow processes, and for 90 days the EDTA-extractable Cd tended to be stable. The decrease in EDTA-extractable Cd may be due to precipitation/nucleation processes, diffusion of Cd into the micropores/mesopores, and occlusion within organic matter in soils. A semi-mechanistic model to predict the extractability of Cd during incubation, based on processes of Cd precipitation/nucleation, diffusion, and occlusion within organic matter, was developed and calibrated. The results showed that the processes of micropore/mesopore diffusion were predominant processes affecting the extractability of Cd added to soils, and were slow. However, the proportions of the processes of precipitation/nucleation and occlusion within organic matter to the non-EDTA-extractable Cd added to soils were only 0.03–21.0% and 0.41–6.95%, respectively. The measured EDTA-extractable Cd from incubated soils were in good agreement with those predicted by the semi-mechanistic model (R2 = 0.829. The results also indicated that soil pH, organic matter, and incubation time were the most important factors affecting Cd aging.

  18. [Application of DNA extraction kit, 'GM quicker' for detection of genetically modified soybeans].

    Science.gov (United States)

    Sato, Noriko; Sugiura, Yoshitsugu; Tanaka, Toshitsugu

    2012-01-01

    Several DNA extraction methods have been officially introduced to detect genetically modified soybeans, but the choice of DNA extraction kits depend on the nature of the samples, such as grains or processed foods. To overcome this disadvantage, we examined whether the GM quicker kit is available for both grains and processed foods. We compared GM quicker with four approved DNA extraction kits in respect of DNA purity, copy numbers of lectin gene, and working time. We found that the DNA quality of GM quicker was superior to that of the other kits for grains, and the procedure was faster. However, in the case of processed foods, GM quicker was not superior to the other kits. We therefore investigated an unapproved GM quicker 3 kit, which is available for DNA extraction from processed foods, such as tofu and boiled soybeans. The GM quicker 3 kit provided good DNA quality from both grains and processed foods, so we made a minor modification of the GM quicker-based protocol that was suitable for processed foods, using GM quicker and its reagents. The modified method enhanced the performance of GM quicker with processed foods. We believe that GM quicker with the modified protocol is an excellent tool to obtain high-quality DNA from grains and processed foods for detection of genetically modified soybeans.

  19. DNA extraction methods for panbacterial and panfungal PCR detection in intraocular fluids.

    Science.gov (United States)

    Mazoteras, Paloma; Bispo, Paulo José Martins; Höfling-Lima, Ana Luisa; Casaroli-Marano, Ricardo P

    2015-07-01

    Three different methods of DNA extraction from intraocular fluids were compared with subsequent detection for bacterial and fungal DNA by universal PCR amplification. Three DNA extraction methods, from aqueous and vitreous humors, were evaluated to compare their relative efficiency. Bacterial (Gram positive and negative) and fungal strains were used in this study: Escherichia coli, Staphylococcus epidermidis and Candida albicans. The quality, quantification, and detection limit for DNA extraction and PCR amplification were analyzed. Validation procedures for 13 aqueous humor and 14 vitreous samples, from 20 patients with clinically suspected endophthalmitis were carried out. The column-based extraction method was the most time-effective, achieving DNA detection limits ≥10(2) and 10(3 )CFU/100 µL for bacteria and fungi, respectively. PCR amplification detected 100 fg, 1 pg and 10 pg of genomic DNA of E. coli, S. epidermidis and C. albicans respectively. PCR detected 90.0% of the causative agents from 27 intraocular samples collected from 20 patients with clinically suspected endophthalmitis, while standard microbiological techniques could detect only 60.0%. The most frequently found organisms were Streptococcus spp. in 38.9% (n = 7) of patients and Staphylococcus spp. found in 22.2% (n = 4). The column-based extraction method for very small inocula in small volume samples (50-100 µL) of aqueous and/or vitreous humors allowed PCR amplification in all samples with sufficient quality for subsequent sequencing and identification of the microorganism in the majority of them.

  20. Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.

    Directory of Open Access Journals (Sweden)

    Shichu Huang

    Full Text Available In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10(-2 pg of C. difficile DNA while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health.

  1. Low Cost Extraction and Isothermal Amplification of DNA for Infectious Diarrhea Diagnosis

    Science.gov (United States)

    Huang, Shichu; Do, Jaephil; Mahalanabis, Madhumita; Fan, Andy; Zhao, Lei; Jepeal, Lisa; Singh, Satish K.; Klapperich, Catherine M.

    2013-01-01

    In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10−2 pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health. PMID:23555883

  2. Accelerated solvent extraction method with one-step clean-up for hydrocarbons in soil

    International Nuclear Information System (INIS)

    Nurul Huda Mamat Ghani; Norashikin Sain; Rozita Osman; Zuraidah Abdullah Munir

    2007-01-01

    The application of accelerated solvent extraction (ASE) using hexane combined with neutral silica gel and sulfuric acid/ silica gel (SA/ SG) to remove impurities prior to analysis by gas chromatograph with flame ionization detector (GC-FID) was studied. The efficiency of extraction was evaluated based on the three hydrocarbons; dodecane, tetradecane and pentadecane spiked to soil sample. The effect of ASE operating conditions (extraction temperature, extraction pressure, static time) was evaluated and the optimized condition obtained from the study was extraction temperature of 160 degree Celsius, extraction pressure of 2000 psi with 5 minutes static extraction time. The developed ASE with one-step clean-up method was applied in the extraction of hydrocarbons from spiked soil and the amount extracted was comparable to ASE extraction without clean-up step with the advantage of obtaining cleaner extract with reduced interferences. Therefore in the developed method, extraction and clean-up for hydrocarbons in soil can be achieved rapidly and efficiently with reduced solvent usage. (author)

  3. Selective extraction of hydrocarbons, phosphonates and phosphonic acids from soils by successive supercritical fluid and pressurized liquid extractions.

    Science.gov (United States)

    Chaudot, X; Tambuté, A; Caude, M

    2000-01-14

    Hydrocarbons, dialkyl alkylphosphonates and alkyl alkylphosphonic acids are selectively extracted from spiked soils by successive implementation of supercritical carbon dioxide, supercritical methanol-modified carbon dioxide and pressurized water. More than 95% of hydrocarbons are extracted during the first step (pure supercritical carbon dioxide extraction) whereas no organophosphorus compound is evidenced in this first extract. A quantitative extraction of phosphonates is achieved during the second step (methanol-modified supercritical carbon dioxide extraction). Polar phosphonic acids are extracted during a third step (pressurized water extraction) and analyzed by gas chromatography under methylated derivatives (diazomethane derivatization). Global recoveries for these compounds are close to 80%, a loss of about 20% occurring during the derivatization process (co-evaporation with solvent). The developed selective extraction method was successfully applied to a soil sample during an international collaborative exercise.

  4. A rapid and efficient DNA extraction protocol from fresh and frozen human blood samples.

    Science.gov (United States)

    Guha, Pokhraj; Das, Avishek; Dutta, Somit; Chaudhuri, Tapas Kumar

    2018-01-01

    Different methods available for extraction of human genomic DNA suffer from one or more drawbacks including low yield, compromised quality, cost, time consumption, use of toxic organic solvents, and many more. Herein, we aimed to develop a method to extract DNA from 500 μL of fresh or frozen human blood. Five hundred microliters of fresh and frozen human blood samples were used for standardization of the extraction procedure. Absorbance at 260 and 280 nm, respectively, (A 260 /A 280 ) were estimated to check the quality and quantity of the extracted DNA sample. Qualitative assessment of the extracted DNA was checked by Polymerase Chain reaction and double digestion of the DNA sample. Our protocol resulted in average yield of 22±2.97 μg and 20.5±3.97 μg from 500 μL of fresh and frozen blood, respectively, which were comparable to many reference protocols and kits. Besides yielding bulk amount of DNA, our protocol is rapid, economical, and avoids toxic organic solvents such as Phenol. Due to unaffected quality, the DNA is suitable for downstream applications. The protocol may also be useful for pursuing basic molecular researches in laboratories having limited funds. © 2017 Wiley Periodicals, Inc.

  5. [DNA extraction from decomposed tissue by double-digest and magnetic beads methods].

    Science.gov (United States)

    Yang, Dian; Liu, Chao; Liu, Hong

    2011-12-01

    To study the effect of the double-digest and magnetic beads method for DNA extraction from 3 types of decomposed tissues. DNA of cartilages, nails and joint capsule in 91 highly decomposed corpses which had not been extracted by common magnetic beads method, were prepared with the double-digest and magnetic beads methods, and quantified with Quantifiler kit, followed by amplification with Sinofiler kit or Minifiler kit. DNA concentration extracted from the 91 highly decomposed cartilages, nails and joint capsule samples was 0-0.225 ng/microL. Sixty-two samples whose DNA concentration were more than 0.020 ng/microL had obtained 9 or more STR loci successfully. The detection rate was 68.13%. The successful rate of STR genotyping for the 3 types of decomposed tissues can be significantly improved by the double-digest and magnetic beads methods.

  6. An effective and low-cost method for DNA extraction from herbal ...

    African Journals Online (AJOL)

    Rhubarb is an important traditional Chinese herbal drug with high secondary metabolites that interfere with DNA extraction procedures and downstream applications, such as DNA restriction and amplification. An effective and low-cost protocol for isolating genomic DNA from root of Rheum tanguticum is described in this ...

  7. Green Remediation Best Management Practices: Soil Vapor Extraction & Air Sparging

    Science.gov (United States)

    Historically, approximately one-quarter of Superfund source control projects have involved soil vapor extraction (SVE) to remove volatile organic compounds (VOCs) sorbed to soil in the unsaturated (vadose) zone.

  8. Extraction of DNA from Forensic Biological Samples for Genotyping.

    Science.gov (United States)

    Stray, J E; Liu, J Y; Brevnov, M G; Shewale, J G

    2010-07-01

    Biological forensic samples constitute evidence with probative organic matter. Evidence believed to contain DNA is typically processed for extraction and purification of its nucleic acid content. Forensic DNA samples are composed of two things, a tissue and the substrate it resides on. Compositionally, a sample may contain almost anything and for each, the type, integrity, and content of both tissue and substrate will vary, as will the contaminant levels. This fact makes the success of extraction one of the most unpredictable steps in genotypic analysis. The development of robust genotyping systems and analysis platforms for short tandem repeat (STR) and mitochondrial DNA sequencing and the acceptance of results generated by these methods in the court system, resulted in a high demand for DNA testing. The increasing variety of sample submissions created a need to isolate DNA from forensic samples that may be compromised or contain low levels of biological material. In the past decade, several robust chemistries and isolation methods have been developed to safely and reliably recover DNA from a wide array of sample types in high yield and free of PCR inhibitors. In addition, high-throughput automated workflows have been developed to meet the demand for processing increasing numbers of samples. This review summarizes a number of the most widely adopted methods and the best practices for DNA isolation from forensic biological samples, including manual, semiautomated, and fully automated platforms. Copyright © 2010 Central Police University.

  9. Dynamics of dissolved and extractable organic nitrogen upon soil amendment with crop residues

    NARCIS (Netherlands)

    Ros, G.H.; Hoffland, E.

    2010-01-01

    Dissolved organic nitrogen (DON) is increasingly recognized as a pivotal pool in the soil nitrogen (N) cycle. Numerous devices and sampling procedures have been used to estimate its size, varying from in situ collection of soil solution to extraction of dried soil with salt solutions. Extractable

  10. Comparison of Three Different DNA Extraction Methods for Linguatula serrata as a Food Born Pathogen

    Directory of Open Access Journals (Sweden)

    Gilda ESLAMI

    2017-06-01

    Full Text Available Background: One of the most important items in molecular characterization of food-borne pathogens is high quality genomic DNA. In this study, we investigated three protocols and compared their simplicity, duration and costs for extracting genomic DNA from Linguatula serrata.Methods: The larvae were collected from the sheep’s visceral organs from the Yazd Slaughterhouse during May 2013. DNA extraction was done in three different methods, including commercial DNA extraction kit, Phenol Chloroform Isoamylalcohol (PCI, and salting out. Extracted DNA in each method was assessed for quantity and quality using spectrophotometery and agarose gel electrophoresis, respectively.Results: The less duration was regarding to commercial DNA extraction kit and then salting out protocol. The cost benefit one was salting out and then PCI method. The best quantity was regarding to PCI with 72.20±29.20 ng/μl, and purity of OD260/OD280 in 1.76±0.947. Agarose gel electrophoresis for assessing the quality found all the same.Conclusion: Salting out is introduced as the best method for DNA extraction from L. seratta as a food-borne pathogen with the least costand appropriate purity. Although, the best purity was regarding to PCI but PCI is not safe as salting out. In addition, the duration of salting out was less than PCI. The least duration was seen in commercial DNA extraction kit, but it is expensive and therefore is not recommended for developing countries where consumption of offal is common.

  11. Evaluation of DNA extraction methods for the detection of Cytomegalovirus in dried blood spots

    Science.gov (United States)

    Koontz, D.; Baecher, K.; Amin, M.; Nikolova, S.; Gallagher, M.; Dollard, S.

    2015-01-01

    Background Dried blood spots (DBS) are collected universally from newborns and may be valuable for the diagnosis of congenital Cytomegalovirus (CMV) infection. The reported analytical sensitivity for DBS testing compared to urine or saliva varies greatly across CMV studies. The purpose of this study was to directly compare the performance of various DNA extraction methods for identification of CMV in DBS including those used most often in CMV studies. Study design Whatman® Grade 903 filter paper cards were spotted with blood samples from 25 organ transplant recipients who had confirmed CMV viremia. Six DNA extraction methods were compared for relative yield of viral and cellular DNA: 2 manual solution-based methods (Gentra Puregene, thermal shock), 2 manual silica column-based methods (QIAamp DNA Mini, QIAamp DNA Investigator), and 2 automated methods (M48 MagAttract Mini, QIAcube Investigator). DBS extractions were performed in triplicate followed by real-time quantitative PCR (qPCR). Results For extraction of both viral and cellular DNA, two methods (QIAamp DNA Investigator and thermal shock) consistently gave the highest yields, and two methods (M48 MagAttract Mini and QIAamp DNA Mini) consistently gave the lowest yields. There was an average 3-fold difference in DNA yield between the highest and lowest yield methods. Conclusion The choice of DNA extraction method is a major factor in the ability to detect low levels of CMV in DBS and can largely account for the wide range of DBS sensitivities reported in studies to date. PMID:25866346

  12. Comparison of two commercial DNA extraction kits for the analysis of nasopharyngeal bacterial communities

    Directory of Open Access Journals (Sweden)

    Keith A. Crandall

    2016-04-01

    Full Text Available Characterization of microbial communities via next-generation sequencing (NGS requires an extraction ofmicrobial DNA. Methodological differences in DNA extraction protocols may bias results and complicate inter-study comparisons. Here we compare the effect of two commonly used commercial kits (Norgen and Qiagenfor the extraction of total DNA on estimatingnasopharyngeal microbiome diversity. The nasopharynxis a reservoir for pathogens associated with respiratory illnesses and a key player in understandingairway microbial dynamics. Total DNA from nasal washes corresponding to 30 asthmatic children was extracted using theQiagenQIAamp DNA and NorgenRNA/DNA Purification kits and analyzed via IlluminaMiSeq16S rRNA V4 ampliconsequencing. The Norgen samples included more sequence reads and OTUs per sample than the Qiagen samples, but OTU counts per sample varied proportionallybetween groups (r = 0.732.Microbial profiles varied slightly between sample pairs, but alpha- and beta-diversity indices (PCoAand clustering showed highsimilarity between Norgen and Qiagenmicrobiomes. Moreover, no significant differences in community structure (PERMANOVA and adonis tests and taxa proportions (Kruskal-Wallis test were observed betweenkits. Finally, aProcrustes analysis also showed low dissimilarity (M2 = 0.173; P< 0.001 between the PCoAs of the two DNA extraction kits. Contrary to what has been observed in previous studies comparing DNA extraction methods, our 16S NGS analysis of nasopharyngeal washes did not reveal significant differences in community composition or structure between kits. Our findingssuggest congruence between column-based chromatography kits and supportthe comparison of microbiomeprofilesacross nasopharyngeal metataxonomic studies.

  13. Evaluation of Sample Stability and Automated DNA Extraction for Fetal Sex Determination Using Cell-Free Fetal DNA in Maternal Plasma

    Directory of Open Access Journals (Sweden)

    Elena Ordoñez

    2013-01-01

    Full Text Available Objective. The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. Methods. A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR. Results. Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL, the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses. Conclusion. We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe.

  14. Subcritical Water Extraction of Amino Acids from Atacama Desert Soils

    Science.gov (United States)

    Amashukeli, Xenia; Pelletier, Christine C.; Kirby, James P.; Grunthaner, Frank J.

    2007-01-01

    Amino acids are considered organic molecular indicators in the search for extant and extinct life in the Solar System. Extraction of these molecules from a particulate solid matrix, such as Martian regolith, will be critical to their in situ detection and analysis. The goals of this study were to optimize a laboratory amino acid extraction protocol by quantitatively measuring the yields of extracted amino acids as a function of liquid water temperature and sample extraction time and to compare the results to the standard HCl vapor- phase hydrolysis yields for the same soil samples. Soil samples from the Yungay region of the Atacama Desert ( Martian regolith analog) were collected during a field study in the summer of 2005. The amino acids ( alanine, aspartic acid, glutamic acid, glycine, serine, and valine) chosen for analysis were present in the samples at concentrations of 1 - 70 parts- per- billion. Subcritical water extraction efficiency was examined over the temperature range of 30 - 325 degrees C, at pressures of 17.2 or 20.0 MPa, and for water- sample contact equilibration times of 0 - 30 min. None of the amino acids were extracted in detectable amounts at 30 degrees C ( at 17.2 MPa), suggesting that amino acids are too strongly bound by the soil matrix to be extracted at such a low temperature. Between 150 degrees C and 250 degrees C ( at 17.2 MPa), the extraction efficiencies of glycine, alanine, and valine were observed to increase with increasing water temperature, consistent with higher solubility at higher temperatures, perhaps due to the decreasing dielectric constant of water. Amino acids were not detected in extracts collected at 325 degrees C ( at 20.0 MPa), probably due to amino acid decomposition at this temperature. The optimal subcritical water extraction conditions for these amino acids from Atacama Desert soils were achieved at 200 degrees C, 17.2 MPa, and a water- sample contact equilibration time of 10 min.

  15. Hydroxypropyl-beta-cyclodextrin as non-exhaustive extractant for organochlorine pesticides and polychlorinated biphenyls in muck soil

    Energy Technology Data Exchange (ETDEWEB)

    Wong, Fiona [Centre for Atmospheric Research Experiments, Science and Technology Branch, Environment Canada, 6248 Eighth Line, Egbert, Ontario, L0L 1N0 (Canada); Department of Chemistry, University of Toronto Scarborough, 1265 Military Trail, Toronto, Ontario, M1C 1A4 (Canada); Bidleman, Terry F., E-mail: terry.bidleman@ec.gc.c [Centre for Atmospheric Research Experiments, Science and Technology Branch, Environment Canada, 6248 Eighth Line, Egbert, Ontario, L0L 1N0 (Canada)

    2010-05-15

    Hydroxypropyl-beta-cyclodextrin (HPCD) was used as a non-exhaustive extractant for organochlorine pesticides (OCs) and polychlorinated biphenyls (PCBs) in muck soil. An optimized extraction method was developed which involved using a HPCD to soil mass ratio of 5.8 with a single extraction period of 20 h. An aging experiment was performed by spiking a muck soil with {sup 13}C-labeled OCs and non-labeled PCBs. The soil was extracted with the optimized HPCD method and Soxhlet apparatus with dichloromethane over 550 d periodically. The HPCD extractability of the spiked OCs was greater than of the native OCs. A decreased in HPCD extractability was observed for the spiked OCs after 550 d of aging and their extractability approached those of the natives. The partition coefficient between HPCD and soil (log K{sub CD-Soil}) was negatively correlated with the octanol-water partition coefficient (log K{sub OW}) with r{sup 2} = 0.67 and p < 0.05. - The effect of aging on the extractability of organochlorine chemicals in muck soil was investigated using hydroxypropyl-beta-cyclodextrin as a mild extractant.

  16. Chemical and plant extractability of metals and plant growth on soils amended with sludge

    Energy Technology Data Exchange (ETDEWEB)

    Gaynor, J.D.; Halstead, R.L.

    1976-02-01

    The addition of sludge to a Fox sandy loam (sl), Granby sl and Rideau clay (c) soil increased soil pH, total C, NaHCO3 extractable P, cation exchange capacity and exchangeable Ca. Sludge application increased DTPA-extractable Cd 2 to 5 times, Pb 2 to 3 times, Cu 3 to 7 times and Zn 7 to 31 times. Metal extractability in Granby and Fox sl soils was not greatly changed after 11 mo incubation but extractable Zn, Cu, Pb and Cd were reduced in the clay soil following incubation. Cropping to lettuce reduced the quantity of metal extracted from Fox sl soil and to a lesser extent from Rideau c soil but not from Granby sl soil. Lettuce (Lactuca sativa L.) yields were significantly reduced for the first crop grown on sludge + fertilizer-treated Rideau c and Granby sl soils and for all three harvests from similarly treated Fox s 1 soil compared to harvests from soils treated with fertilizer only. Yield reduction for the first crop was attributed to a salt effect, as subsequent yields on Rideau c and Granby sl soils were similar to harvests from fertilized treatments. Saturation extract conductivities for all sludge treatments were higher for incubated than for cropped soils. Generally Zn, Cu and Pb tissue concentrations in lettuce harvested from sludge + fertilizer-treated Fox and Granby sl soils were significantly increased but total uptake was only increased for Zn. Metal uptake and tissue concentrations for lettuce grown on similarly treated Rideau c soil were equal to or less than those found in lettuce harvested from the fertilizer-only treatment. To a lesser extent similar trends were observed with the tomato (Lycospersicon esculentum Mill.) crop. 27 references, 3 tables.

  17. A RAPID DNA EXTRACTION METHOD FOR PCR IDENTIFICATION OF FUNGAL INDOOR AIR CONTAMINANTS

    Science.gov (United States)

    Following air sampling, fungal DNA needs to be extracted and purified to a state suitable for laboratory use. Our laboratory has developed a simple method of extraction and purification of fungal DNA appropriate for enzymatic manipulation and polymerase chain reaction (PCR) appli...

  18. Comparison of mild extraction procedures for determination of plant-available arsenic compounds in soil

    Energy Technology Data Exchange (ETDEWEB)

    Szakova, Jirina; Tlustos, Pavel; Pavlikova, Daniela; Balik, Jiri [Czech University of Agriculture, Department of Agrochemistry and Plant Nutrition, Prague (Czech Republic); Goessler, Walter; Schlagenhaufen, Claudia [Karl-Franzens-University Graz, Institute of Chemistry, Analytical Chemistry, Graz (Austria)

    2005-05-01

    In this work three mild extraction agents for determination of plant-available fractions of elements in soil were evaluated for arsenic speciation in soil samples. Pepper (Capsicum annum, L.) var. California Wonder was cultivated in pots, and aqueous solutions of arsenite, arsenate, methylarsonic acid, and dimethylarsinic acid, at a concentration of 15 mg As kg{sup -1} soil, were added at the beginning of the experiment. Control pots (untreated) were also included. Deionized water, 0.01 mol L{sup -1} CaCl{sub 2}, and 0.05 mol L{sup -1} (NH{sub 4}){sub 2}SO{sub 4} were used to extract the plant-available fraction of the arsenic compounds in soil samples collected during the vegetation period of the plants. Whereas in control samples the extractable arsenic fraction did not exceed 1% of total arsenic content, soil amendment by arsenic compounds resulted in extraction of larger amounts, which varied between 1.4 and 8.1% of total arsenic content, depending on soil treatment and on the extracting agent applied. Among arsenic compounds determined by HPLC-ICPMS arsenate was predominant, followed by small amounts of arsenite, methylarsonic acid, and dimethylarsinic acid, depending on the individual soil treatment. In all the experiments in which methylarsonic acid was added to the soil methylarsonous acid was detected in the extracts, suggesting that the soil bacteria are capable of reducing methylarsonic acid before a further methylation occurs. No significant differences were observed between analytical data obtained by using different extraction procedures. (orig.)

  19. The effects of three different grinding methods in DNA extraction of ...

    African Journals Online (AJOL)

    Rapid DNA extraction is a prerequisite for molecular studies. Generally, plant tissue is ground in liquid nitrogen to isolate DNA; but, liquid nitrogen is dangerous and volatile. Besides, liquid nitrogen is not always available in many developing countries. To investigate if high quality DNA could be obtained for downstream ...

  20. [Study on a collagenase protocol to extract DNA from remnant feathers in edible bird's nest].

    Science.gov (United States)

    Wang, Ling-Li; Chen, Nian; Zhang, Wei-Wei; Wu, Guo-Hong; Lai, Xiao-Ping

    2013-08-01

    To establish a method for extracting genomic DNA from rudimental bird feather from the precious edible bird's nest (EBN) harvested from the swiftlet cave. Observed the EBN using endoscopic and studied the influence of adding collagenase on the extracting yield of DNA. PCR amplification and sequencing for the extraction was also conducted. Collagenase was used in addition to protease K which could substantively increase the DNA yield. The DNA extracted by this method could be used for PCR and other molecular biology analyses. This method can be applied to identify the species types in biological products, especially for animal tissue materials that rich in collagen.

  1. Remediation of lead-contaminated soil with non-toxic biodegradable natural ligands extracted from soybean.

    Science.gov (United States)

    Lee, Yong-Woo; Kim, Chulsung

    2012-01-01

    Bench-scale soil washing studies were performed to evaluate the potential application of non-toxic, biodegradable extracted soybean-complexing ligands for the remediation of lead-contaminated soils. Results showed that, with extracted soybean-complexing ligands, lead solubility extensively increased when pH of the solution was higher than 6, and approximately 10% (500 mg/kg) of lead was removed from a rifle range soil. Two potential primary factors controlling the effectiveness of lead extraction from lead-contaminated soils with natural ligands are adsorption of extracted aqueous lead ions onto the ground soybean and the pH of the extraction solution. More complexing ligands were extracted from the ground soybean as the reaction pH increased. As a result, significantly higher lead extraction efficiency was observed under basic environments. In addition, less adsorption onto soybean was observed when the pH of the solution was higher than 7. Among two available Lewis base functional groups in the extracted soybean-complexing ligands such as carboxylate and the alpha-amino functional groups, the non-protonated alpha-amino functional groups may play an important role for the dissolution of lead from lead-contaminated soil through the formation of soluble lead--ligand complexes.

  2. Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method.

    Science.gov (United States)

    McFall, Sally M; Neto, Mário F; Reed, Jennifer L; Wagner, Robin L

    2016-08-06

    FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 µl whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

  3. How to open the treasure chest? Optimising DNA extraction from herbarium specimens.

    Science.gov (United States)

    Särkinen, Tiina; Staats, Martijn; Richardson, James E; Cowan, Robyn S; Bakker, Freek T

    2012-01-01

    Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance in terms of DNA purity and yield, and PCR amplification success as measured by using three differentially sized markers, the rbcL barcoding marker (cpDNA), the LEAFY exon 3 (nrDNA), and the trnL((UAA)) P6 loop (cpDNA). Results reveal large differences between extraction methods, where DNA purity rather than yield is shown to be strongly correlated with PCR success. Amplicon size shows similarly strong correlation with PCR success, with the shortest fragment showing the highest success rate (78%, P6 loop, 10-143 base pairs (bp)) and the largest fragment the lowest success (10%, rbcL, 670 bp). The effect of specimen preparation method on PCR success was also tested. Results show that drying method strongly affects PCR success, especially the availability of fragments longer than 250 bp, where longer fragments are more available for PCR amplification in air dried material compared to alcohol dried specimens. Results from our study indicate that projects relying on poor-quality starting material such as herbarium or scat samples should focus on extracting pure DNA and aim to amplify short target regions (herbarium samples available into barcoding initiatives and other molecular studies.

  4. Nonhomologous DNA End Joining in Cell-Free Extracts

    Directory of Open Access Journals (Sweden)

    Sheetal Sharma

    2010-01-01

    Full Text Available Among various DNA damages, double-strand breaks (DSBs are considered as most deleterious, as they may lead to chromosomal rearrangements and cancer when unrepaired. Nonhomologous DNA end joining (NHEJ is one of the major DSB repair pathways in higher organisms. A large number of studies on NHEJ are based on in vitro systems using cell-free extracts. In this paper, we summarize the studies on NHEJ performed by various groups in different cell-free repair systems.

  5. A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains.

    Science.gov (United States)

    Zhang, Y J; Zhang, S; Liu, X Z; Wen, H A; Wang, M

    2010-07-01

    A simple and rapid method (designated thermolysis) for extracting genomic DNA from bulk fungal strains was described. In the thermolysis method, a few mycelia or yeast cells were first rinsed with pure water to remove potential PCR inhibitors and then incubated in a lysis buffer at 85 degrees C to break down cell walls and membranes. This method was used to extract genomic DNA from large numbers of fungal strains (more than 92 species, 35 genera of three phyla) isolated from different sections of natural Ophiocordyceps sinensis specimens. Regions of interest from high as well as single-copy number genes were successfully amplified from the extracted DNA samples. The DNA samples obtained by this method can be stored at -20 degrees C for over 1 year. The method was effective, easy and fast and allowed batch DNA extraction from multiple fungal isolates. Use of the thermolysis method will allow researchers to obtain DNA from fungi quickly for use in molecular assays. This method requires only minute quantities of starting material and is suitable for diverse fungal species.

  6. A Suitable Method for DNA Extraction from Bones for Forensic Applications: A Case Study

    Directory of Open Access Journals (Sweden)

    Aqeela S. Abuidrees

    2016-06-01

    Full Text Available Human identification techniques are constantly developing. Before the discovery of DNA, anthropology accompanied with odontology was the most applicable technique for human identification. With the new era of molecular biology and the revolution of DNA and PCR techniques, DNA profiling has become the core of the human forensic identification process. Different types of samples can be exploited in forensic DNA analysis. In some extreme cases, bone samples are the only accessible samples of DNA due to the bad conditions of putrefaction or degradation of other biological materials and tissues. Therefore, an appropriate method should be determined to yield a full and clean profile. A case study is presented here in order to identify human remains and conclude the most appropriate method of DNA extraction from human remains. In addition, this study looks at the best part of the skeletal remains to be considered in the extraction of DNA for the purposes of identification. A suspect admitted that he buried his aborted son six months ago. The remains were recovered and DNA analysis was performed in order to determine any genetic link of the remains to the suspect and the female who delivered the baby. Two extraction methods were compared, the standard organic (phenol:chloroform:isoamyl alcohol and automated extraction using magnetic beads coated with silica (Qiagen EZ1 Advanced XL. Two bone parts, femur and clavicle, were also compared in terms of DNA yield. The efficiency of the two methods of DNA extraction from bones is illustrated quantitatively and qualitatively. Paternity testing was performed and the suspect was excluded from being the alleged father.

  7. Rapid and efficient extraction of genomic DNA from different phytopathogenic fungi using DNAzol reagent.

    Science.gov (United States)

    Guo, Jian-Rong; Schnieder, F; Abd-Elsalam, K A; Verreet, J A

    2005-01-01

    A modified procedure using the commercial DNAzol reagent was successfully applied to extract genomic DNA from 25 fungal species. The DNA yield varied from 306 to 1,927 microg g(-1) dry mycelia and the A(260)/A(280) ratio from 1.59 to 1.93. Compared with the method of J.L. Cenis (Nucleic Acids Res. 1992, 20: 2380) this procedure generated a higher DNA yield from 17 species and a higher A(260)/A(280) ratio from 23 species. But for four species, Cenis (1992) method was more suitable. No inhibitor of polymerase chain reaction was evident for the DNA extracted by the modified procedure, whereas some inhibitors remained in DNA of eight species extracted by the previous method.

  8. Subcritical water extraction of amino acids from Mars analog soils.

    Science.gov (United States)

    Noell, Aaron C; Fisher, Anita M; Fors-Francis, Kisa; Sherrit, Stewart

    2018-01-18

    For decades, the Martian regolith has stymied robotic mission efforts to catalog the organic molecules present. Perchlorate salts, found widely throughout Mars, are the main culprit as they breakdown and react with organics liberated from the regolith during pyrolysis, the primary extraction technique attempted to date on Mars. This work further develops subcritical water extraction (SCWE) as a technique for extraction of amino acids on future missions. The effect of SCWE temperature (185, 200, and 215°C) and duration of extraction (10-120 min) on the total amount and distribution of amino acids recovered was explored for three Mars analog soils (JSC Mars-1A simulant, an Atacama desert soil, and an Antarctic Dry Valleys soil) and bovine serum albumin (as a control solution of known amino acid content). Total amounts of amino acids extracted increased with both time and temperature; however, the distribution shifted notably due to the destruction of the amino acids with charged or polar side chains at the higher temperatures. The pure bovine serum albumin solution and JSC Mars 1A also showed lower yields than the Atacama and Antarctic extractions suggesting that SCWE may be less effective at hydrolyzing large or aggregated proteins. Changing solvent from water to a dilute (10 mM) HCl solution allowed total extraction efficiencies comparable to the higher temperature/time combinations while using the lowest temperature/time (185°C/20 min). The dilute HCl extractions also did not lead to the shift in amino acid distribution observed at the higher temperatures. Additionally, adding sodium perchlorate salt to the extraction did not interfere with recoveries. Native magnetite in the JSC Mars-1A may have been responsible for destruction of glycine, as evidenced by its uncharacteristic decrease as the temperature/time of extraction increased. This work shows that SCWE can extract high yields of native amino acids out of Mars analog soils with minimal disruption of the

  9. Analysis of perfluorinated carboxylic acids in soils II: optimization of chromatography and extraction.

    Science.gov (United States)

    Washington, John W; Henderson, W Matthew; Ellington, J Jackson; Jenkins, Thomas M; Evans, John J

    2008-02-15

    With the objective of detecting and quantitating low concentrations of perfluorinated carboxylic acids (PFCAs), including perfluorooctanoic acid (PFOA), in soils, we compared the analytical suitability of liquid chromatography columns containing three different stationary phases, two different liquid chromatography-tandem mass spectrometry (LC/MS/MS) systems, and eight combinations of sample-extract pretreatments, extractions and cleanups on three test soils. For the columns and systems we tested, we achieved the greatest analytical sensitivity for PFCAs using a column with a C(18) stationary phase in a Waters LC/MS/MS. In this system we achieved an instrument detection limit for PFOA of 270 ag/microL, equating to about 14 fg of PFOA on-column. While an elementary acetonitrile/water extraction of soils recovers PFCAs effectively, natural soil organic matter also dissolved in the extracts commonly imparts significant noise that appears as broad, multi-nodal, asymmetric peaks that coelute with several PFCAs. The intensity and elution profile of this noise is highly variable among soils and it challenges detection of low concentrations of PFCAs by decreasing the signal-to-noise contrast. In an effort to decrease this background noise, we investigated several methods of pretreatment, extraction and cleanup, in a variety of combinations, that used alkaline and unbuffered water, acetonitrile, tetrabutylammonium hydrogen sulfate, methyl-tert-butyl ether, dispersed activated carbon and solid-phase extraction. For the combined objectives of complete recovery and minimization of background noise, we have chosen: (1) alkaline pretreatment; (2) extraction with acetonitrile/water; (3) evaporation to dryness; (4) reconstitution with tetrabutylammonium-hydrogen-sulfate ion-pairing solution; (5) ion-pair extraction to methyl-tert-butyl ether; (6) evaporation to dryness; (7) reconstitution with 60/40 acetonitrile/water (v/v); and (8) analysis by LC/MS/MS. Using this method, we

  10. An improved method of DNA extraction from plants for pathogen ...

    African Journals Online (AJOL)

    Polymerase chain reaction (PCR)-based applications in plant molecular biology and molecular diagnostics for plant pathogens require good quality DNA for reliable and reproducible results. Leaf tissue is often the choice for DNA extraction, but the use of other sources such as tubers, stems, or seeds, is not uncommon.

  11. Rapid extraction of genomic DNA from medically important yeasts and filamentous fungi by high-speed cell disruption.

    Science.gov (United States)

    Müller, F M; Werner, K E; Kasai, M; Francesconi, A; Chanock, S J; Walsh, T J

    1998-06-01

    Current methods of DNA extraction from different fungal pathogens are often time-consuming and require the use of toxic chemicals. DNA isolation from some fungal organisms is difficult due to cell walls or capsules that are not readily susceptible to lysis. We therefore investigated a new and rapid DNA isolation method using high-speed cell disruption (HSCD) incorporating chaotropic reagents and lysing matrices in comparison to standard phenol-chloroform (PC) extraction protocols for isolation of DNA from three medically important yeasts (Candida albicans, Cryptococcus neoformans, and Trichosporon beigelii) and two filamentous fungi (Aspergillus fumigatus and Fusarium solani). Additional extractions by HSCD were performed on Saccharomyces cerevisiae, Pseudallescheria boydii, and Rhizopus arrhizus. Two different inocula (10(8) and 10(7) CFU) were compared for optimization of obtained yields. The entire extraction procedure was performed on as many as 12 samples within 1 h compared to 6 h for PC extraction. In comparison to the PC procedure, HSCD DNA extraction demonstrated significantly greater yields for 10(8) CFU of C. albicans, T. beigelii, A. fumigatus, and F. solani (P extraction and PC extraction. For 10(7) CFU of T. beigelii, PC extraction resulted in a greater yield than did HSCD (P fungi than for yeasts by the HSCD extraction procedure (P extraction procedure, differences were not significant. For all eight organisms, the rapid extraction procedure resulted in good yield, integrity, and quality of DNA as demonstrated by restriction fragment length polymorphism, PCR, and random amplified polymorphic DNA. We conclude that mechanical disruption of fungal cells by HSCD is a safe, rapid, and efficient procedure for extracting genomic DNA from medically important yeasts and especially from filamentous fungi.

  12. Diverse gene functions in a soil mobilome

    DEFF Research Database (Denmark)

    Luo, Wenting; Xu, Zhuofei; Riber, Leise

    2016-01-01

    Accessing bacterial mobilomes of any given environment enables the investigation of genetic traits encoded by circular genetic elements, and how their transfer drives the adaptation of microbial communities. Here we take advantage of Illumina HiSeq sequencing and report, for the first time......, the soil mobilome sampled from a well-characterized field in Hygum, Denmark. Soil bacterial cells were obtained by Nycodenz extraction, total DNA was purified by removing sheared chromosomal DNA using exonuclease digestion, and the remaining circular DNA was amplified with the phi29 polymerase and finally...... sequenced. The soil mobilome represented a wide range of known bacterial gene functions and highlighted the enrichment of plasmids, transposable elements and phages when compared to a well-characterized soil metagenome that, on the other hand, was dominated by basic biosynthesis and metabolism functions...

  13. MICHIGAN SOIL VAPOR EXTRACTION REMEDIATION (MISER) MODEL: A COMPUTER PROGRAM TO MODEL SOIL VAPOR EXTRACTION AND BIOVENTING OF ORGANIC CHEMICALS IN UNSATURATED GEOLOGICAL MATERIAL

    Science.gov (United States)

    Soil vapor extraction (SVE) and bioventing (BV) are proven strategies for remediation of unsaturated zone soils. Mathematical models are powerful tools that can be used to integrate and quantify the interaction of physical, chemical, and biological processes occurring in field sc...

  14. Impact of Soil Texture on Soil Ciliate Communities

    Science.gov (United States)

    Chau, J. F.; Brown, S.; Habtom, E.; Brinson, F.; Epps, M.; Scott, R.

    2014-12-01

    Soil water content and connectivity strongly influence microbial activities in soil, controlling access to nutrients and electron acceptors, and mediating interactions between microbes within and between trophic levels. These interactions occur at or below the pore scale, and are influenced by soil texture and structure, which determine the microscale architecture of soil pores. Soil protozoa are relatively understudied, especially given the strong control they exert on bacterial communities through predation. Here, ciliate communities in soils of contrasting textures were investigated. Two ciliate-specific primer sets targeting the 18S rRNA gene were used to amplify DNA extracted from eight soil samples collected from Sumter National Forest in western South Carolina. Primer sets 121F-384F-1147R (semi-nested) and 315F-959R were used to amplify soil ciliate DNA via polymerase chain reaction (PCR), and the resulting PCR products were analyzed by gel electrophoresis to obtain quantity and band size. Approximately two hundred ciliate 18S rRNA sequences were obtained were obtained from each of two contrasting soils. Sequences were aligned against the NCBI GenBank database for identification, and the taxonomic classification of best-matched sequences was determined. The ultimate goal of the work is to quantify changes in the ciliate community under short-timescale changes in hydrologic conditions for varying soil textures, elucidating dynamic responses to desiccation stress in major soil ciliate taxa.

  15. DNA Damage Protecting Activity and Antioxidant Potential of Launaea taraxacifolia Leaves Extract.

    Science.gov (United States)

    Adinortey, Michael Buenor; Ansah, Charles; Weremfo, Alexander; Adinortey, Cynthia Ayefoumi; Adukpo, Genevieve Etornam; Ameyaw, Elvis Ofori; Nyarko, Alexander Kwadwo

    2018-01-01

    The leaf extract of Launaea taraxacifolia commonly known as African Lettuce is used locally to treat dyslipidemia and liver diseases, which are associated with oxidative stress. Methanol extract from L. taraxacifolia leaves was tested for its antioxidant activity and its ability to protect DNA from oxidative damage. In vitro antioxidant potential of the leaf extract was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitric oxide (NO), and hydroxyl (OH) radical scavenging assays. Ferric reducing power, total antioxidant capacity (TAC), metal chelating, and anti-lipid peroxidation ability of the extract were also examined using gallic acid, ascorbic acid, citric acid, and ethylenediaminetetraacetic acid as standards. L. taraxacifolia leaves extract showed antioxidant activity with IC 50 values of 16.18 μg/ml (DPPH), 123.3 μg/ml (NO), 128.2 μg/ml (OH radical), 97.94 μg/ml (metal chelating), 80.28 μg/ml (TAC), and 23 μg/ml (anti-lipid peroxidation activity). L. taraxacifolia leaves extract exhibited a strong capability for DNA damage protection at 20 mg/ml concentration. These findings suggest that the methanolic leaf extract of L. taraxacifolia could be used as a natural antioxidant and also as a preventive therapy against diseases such as arteriosclerosis associated with DNA damage.

  16. The variability of standard artificial soils: Behaviour, extractability and bioavailability of organic pollutants

    International Nuclear Information System (INIS)

    Hofman, Jakub; Hovorková, Ivana; Semple, Kirk T.

    2014-01-01

    Highlights: • Artificial soils from different laboratories revealed different fates, behaviour and bioavailability of lindane and phenanthrene. • Lindane behaviour was related to organic carbon. • Phenanthrene behaviour was significantly affected by degrading microorganisms from peat. • Sterilization of artificial soils might reduce unwanted variability. -- Abstract: Artificial soil is an important standard medium and reference material for soil ecotoxicity bioassays. Recent studies have documented the significant variability of their basic properties among different laboratories. Our study investigated (i) the variability of ten artificial soils from different laboratories by means of the fate, extractability and bioavailability of phenanthrene and lindane, and (ii) the relationships of these results to soil properties and ageing. Soils were spiked with 14 C-phenanthrene and 14 C-lindane, and the total residues, fractions extractable by hydroxypropyl-β-cyclodextrin, and the fractions of phenanthrene mineralizable by bacteria were determined after 1, 14, 28 and 56 days. Significant temporal changes in total residues and extractable and mineralizable fractions were observed for phenanthrene, resulting in large differences between soils after 56 days. Phenanthrene mineralization by indigenous peat microorganisms was suggested as the main driver of that, outweighing the effects of organic matter. Lindane total residues and extractability displayed much smaller changes over time and smaller differences between soils related to organic matter. Roughly estimated, the variability between the artificial soils was comparable to natural soils. The implications of such variability for the results of toxicity tests and risk assessment decisions should be identified. We also suggested that the sterilization of artificial soils might reduce unwanted variability

  17. The variability of standard artificial soils: Behaviour, extractability and bioavailability of organic pollutants

    Energy Technology Data Exchange (ETDEWEB)

    Hofman, Jakub, E-mail: hofman@recetox.muni.cz [Research Centre for Toxic Compounds in the Environment (RECETOX), Faculty of Science, Masaryk University, Kamenice 753/5, Brno CZ-62500 (Czech Republic); Hovorková, Ivana [Research Centre for Toxic Compounds in the Environment (RECETOX), Faculty of Science, Masaryk University, Kamenice 753/5, Brno CZ-62500 (Czech Republic); Semple, Kirk T. [Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ (United Kingdom)

    2014-01-15

    Highlights: • Artificial soils from different laboratories revealed different fates, behaviour and bioavailability of lindane and phenanthrene. • Lindane behaviour was related to organic carbon. • Phenanthrene behaviour was significantly affected by degrading microorganisms from peat. • Sterilization of artificial soils might reduce unwanted variability. -- Abstract: Artificial soil is an important standard medium and reference material for soil ecotoxicity bioassays. Recent studies have documented the significant variability of their basic properties among different laboratories. Our study investigated (i) the variability of ten artificial soils from different laboratories by means of the fate, extractability and bioavailability of phenanthrene and lindane, and (ii) the relationships of these results to soil properties and ageing. Soils were spiked with {sup 14}C-phenanthrene and {sup 14}C-lindane, and the total residues, fractions extractable by hydroxypropyl-β-cyclodextrin, and the fractions of phenanthrene mineralizable by bacteria were determined after 1, 14, 28 and 56 days. Significant temporal changes in total residues and extractable and mineralizable fractions were observed for phenanthrene, resulting in large differences between soils after 56 days. Phenanthrene mineralization by indigenous peat microorganisms was suggested as the main driver of that, outweighing the effects of organic matter. Lindane total residues and extractability displayed much smaller changes over time and smaller differences between soils related to organic matter. Roughly estimated, the variability between the artificial soils was comparable to natural soils. The implications of such variability for the results of toxicity tests and risk assessment decisions should be identified. We also suggested that the sterilization of artificial soils might reduce unwanted variability.

  18. A fully continuous supercritical fluid extraction system for contaminated soil

    International Nuclear Information System (INIS)

    Ryan, M.; Stiver, W.H.

    2007-01-01

    Brownfield sites are contaminated sites in an urban setting. There are hundreds of thousands of such sites, where contaminants migrate to the atmosphere, seep into groundwater, runoff into surface water and enter the food chain through plant uptake and soil ingestion. The Sydney Tar Ponds alone contain more than a million tonnes of contaminated soils and sediments. Soil vapour extraction, incineration, bioremediation, solvent extraction and land filling are among the remediation techniques that have been developed for brownfield sites over the years. However, no single technology is ideally suited to all cases because of the diversity of contaminants and diversity of site characterization. This paper focused on supercritical fluid extraction (SFE) which is well suited to sites contaminated with polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and heavy metal. A fully continuous laboratory-scale SFE process for a slurry-based system was designed and constructed to handle the supercritical carbon dioxide (SC-CO 2 ) and the soil slurry. The system continuously pumps carbon dioxide under supercritical conditions and soil slurry into a counter-current contacting column. The testing soil was Delhi loamy sand, spiked with 10 mg/g of naphthalene. The soil slurry ranged from 0.0028 g dry soil per g slurry to 0.072 g/g. The operating temperature was 43 degrees C and the operating pressure was 7.7 MPa. Near steady state, fully continuous flow was achieved with runs lasting up to 2 hours. The quantifiable recoveries of naphthalene from the soil slurry was demonstrated and the mass transfer coefficients for the system were quantified in order to provide the foundation to advance to a full-scale system and costing analysis. 14 refs., 1 tab., 3 figs

  19. A fully continuous supercritical fluid extraction system for contaminated soil

    Energy Technology Data Exchange (ETDEWEB)

    Ryan, M.; Stiver, W.H. [Guelph Univ., ON (Canada). School of Engineering

    2007-04-15

    Brownfield sites are contaminated sites in an urban setting. There are hundreds of thousands of such sites, where contaminants migrate to the atmosphere, seep into groundwater, runoff into surface water and enter the food chain through plant uptake and soil ingestion. The Sydney Tar Ponds alone contain more than a million tonnes of contaminated soils and sediments. Soil vapour extraction, incineration, bioremediation, solvent extraction and land filling are among the remediation techniques that have been developed for brownfield sites over the years. However, no single technology is ideally suited to all cases because of the diversity of contaminants and diversity of site characterization. This paper focused on supercritical fluid extraction (SFE) which is well suited to sites contaminated with polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and heavy metal. A fully continuous laboratory-scale SFE process for a slurry-based system was designed and constructed to handle the supercritical carbon dioxide (SC-CO{sub 2}) and the soil slurry. The system continuously pumps carbon dioxide under supercritical conditions and soil slurry into a counter-current contacting column. The testing soil was Delhi loamy sand, spiked with 10 mg/g of naphthalene. The soil slurry ranged from 0.0028 g dry soil per g slurry to 0.072 g/g. The operating temperature was 43 degrees C and the operating pressure was 7.7 MPa. Near steady state, fully continuous flow was achieved with runs lasting up to 2 hours. The quantifiable recoveries of naphthalene from the soil slurry was demonstrated and the mass transfer coefficients for the system were quantified in order to provide the foundation to advance to a full-scale system and costing analysis. 14 refs., 1 tab., 3 figs.

  20. The use of genus-specific amplicon pyrosequencing to assess phytophthora species diversity using eDNA from soil and water in Northern Spain.

    Science.gov (United States)

    Català, Santiago; Pérez-Sierra, Ana; Abad-Campos, Paloma

    2015-01-01

    Phytophthora is one of the most important and aggressive plant pathogenic genera in agriculture and forestry. Early detection and identification of its pathways of infection and spread are of high importance to minimize the threat they pose to natural ecosystems. eDNA was extracted from soil and water from forests and plantations in the north of Spain. Phytophthora-specific primers were adapted for use in high-throughput Sequencing (HTS). Primers were tested in a control reaction containing eight Phytophthora species and applied to water and soil eDNA samples from northern Spain. Different score coverage threshold values were tested for optimal Phytophthora species separation in a custom-curated database and in the control reaction. Clustering at 99% was the optimal criteria to separate most of the Phytophthora species. Multiple Molecular Operational Taxonomic Units (MOTUs) corresponding to 36 distinct Phytophthora species were amplified in the environmental samples. Pyrosequencing of amplicons from soil samples revealed low Phytophthora diversity (13 species) in comparison with the 35 species detected in water samples. Thirteen of the MOTUs detected in rivers and streams showed no close match to sequences in international sequence databases, revealing that eDNA pyrosequencing is a useful strategy to assess Phytophthora species diversity in natural ecosystems.

  1. Remediation of soils contaminated with particulate depleted uranium by multi stage chemical extraction

    Energy Technology Data Exchange (ETDEWEB)

    Crean, Daniel E. [Immobilisation Science Laboratory, Department of Materials Science and Engineering, The University of Sheffield (United Kingdom); Centre for Radiochemistry Research, School of Chemistry, The University of Manchester (United Kingdom); Livens, Francis R.; Sajih, Mustafa [Centre for Radiochemistry Research, School of Chemistry, The University of Manchester (United Kingdom); Stennett, Martin C. [Immobilisation Science Laboratory, Department of Materials Science and Engineering, The University of Sheffield (United Kingdom); Grolimund, Daniel; Borca, Camelia N. [Swiss Light Source, Paul Scherrer Institute, Villigen (Switzerland); Hyatt, Neil C., E-mail: n.c.hyatt@sheffield.ac.uk [Immobilisation Science Laboratory, Department of Materials Science and Engineering, The University of Sheffield (United Kingdom)

    2013-12-15

    Highlights: • Batch leaching was examined to remediate soils contaminated with munitions depleted uranium. • Site specific maximum extraction was 42–50% total U in single batch with NH{sub 4}HCO{sub 3}. • Analysis of residues revealed partial leaching and secondary carbonate phases. • Sequential batch leaching alternating between NH{sub 4}HCO{sub 3} and citric acid was designed. • Site specific extraction was increased to 68–87% total U in three batch steps. -- Abstract: Contamination of soils with depleted uranium (DU) from munitions firing occurs in conflict zones and at test firing sites. This study reports the development of a chemical extraction methodology for remediation of soils contaminated with particulate DU. Uranium phases in soils from two sites at a UK firing range, MOD Eskmeals, were characterised by electron microscopy and sequential extraction. Uranium rich particles with characteristic spherical morphologies were observed in soils, consistent with other instances of DU munitions contamination. Batch extraction efficiencies for aqueous ammonium bicarbonate (42–50% total DU extracted), citric acid (30–42% total DU) and sulphuric acid (13–19% total DU) were evaluated. Characterisation of residues from bicarbonate-treated soils by synchrotron microfocus X-ray diffraction and X-ray absorption spectroscopy revealed partially leached U(IV)-oxide particles and some secondary uranyl-carbonate phases. Based on these data, a multi-stage extraction scheme was developed utilising leaching in ammonium bicarbonate followed by citric acid to dissolve secondary carbonate species. Site specific U extraction was improved to 68–87% total U by the application of this methodology, potentially providing a route to efficient DU decontamination using low cost, environmentally compatible reagents.

  2. Evaluation of two methods DNA extraction from formalin-fixed, paraffin-embedded tissues on non-optimal conditions

    International Nuclear Information System (INIS)

    Bustamante, Javier Andres; Astudillo, Miryam; Pazos, Alvaro Jairo; Bravo, Luis Eduardo

    2011-01-01

    Paraffin wax embedded tissues are an invaluable material for retrospective studies requiring the application of molecular analysis. Multiple methods are available to extract DNA from these kinds of samples. However, the most common methods are slow and the reagents often contribute to the fragmentation of genetic material. In order to optimize the procedure, two methods for DNA extraction from paraffin embedded tissue non-optimal conditions were used. 47 blocks containing paraffin-embedded biopsies of pleura, lung and pericardium from 24 patients (66.6% males) older than 18 years, with biopsy proven chronic granulomatous inflammation referred to the department of pathology at University Hospital of Valle between 2002 and 2007 were selected. Each sample was subjected to 10 cuts and was to two methods of DNA extraction: 1. conventional and 2. QIAamp - DNA mini kit. The efficiency of the extracted DNA was assessed by spectrophotometry and PCR amplification of a fragment of the housekeeping gene GAPDH. The concentration of DNA samples extracted by the conventional method was of 65.52 ng/Mu l ± 11.47 (mean ± SE) and the 260/280 absorbance ratio ranged between 0.52 and 2.30 the average concentration of DNA of the samples extracted by the commercial method was 60.89 ng/Mu l ± 6.02 (mean ± SE), with an absorbance that fluctuated between 0 and 2.64. The DNA obtained was amplified by PCR, of 47 samples extracted by methods, 25 and 23 respectively the GAPDH gene amplified successfully. The methods used to obtain DNA showed similar performance, highlighting the potential utility of both extraction methods for the retrospective studies from paraffin embedded tissues in unsuitable conditions.

  3. Effect of decreasing acidity on the extractability of inorganic soil phosphorus

    Directory of Open Access Journals (Sweden)

    Helinä Hartikainen

    1981-01-01

    Full Text Available The extractability of P by the water and anion exchange resin methods and reactions of soil inorganic P were investigated with seven acid mineral soil samples incubated with KOH solutions of various concentrations. The results were compared with the analytical data obtained from three soil samples incubated in a prolonged liming experiment. The resin extraction method proved more effective than the water extraction method. The amounts of P desorbed by both methods seemed to increase exponentially as the pH in the soil suspensions rose. The factors involved were discussed. On the basis of fractionation analyses P reacting to changes in the pH and participating in desorption processes was supposed to originate from secondary NH4F and NaOH soluble reserves. In general, as the acidity decreased NH4F-P increased at the expense of NaOH-P. In heavily limed gyttja soil also H2SO4-P increased. This was possibly induced by the precipitation of mobilized P as a Ca compound. The significance of pH in the extractability of soil P seemed somewhat to lessen as the amount of secondary P increased. The results were in accordance with the conception that liming improves the availability of inorganic P to plants and reduces the need for P fertilization. However, increasing of the soil pH involves the risk that P is more easily desorbed to the recipient water by the eroded soil material carried into the watercourse. Therefore, intensive liming is not recommendable close to the shoreline. Further, it should be taken into account that liming of lakes may also result in eutrophication as desorption of sedimentary inorganic P is enhanced.

  4. Remediation of soils contaminated with particulate depleted uranium by multi stage chemical extraction.

    Science.gov (United States)

    Crean, Daniel E; Livens, Francis R; Sajih, Mustafa; Stennett, Martin C; Grolimund, Daniel; Borca, Camelia N; Hyatt, Neil C

    2013-12-15

    Contamination of soils with depleted uranium (DU) from munitions firing occurs in conflict zones and at test firing sites. This study reports the development of a chemical extraction methodology for remediation of soils contaminated with particulate DU. Uranium phases in soils from two sites at a UK firing range, MOD Eskmeals, were characterised by electron microscopy and sequential extraction. Uranium rich particles with characteristic spherical morphologies were observed in soils, consistent with other instances of DU munitions contamination. Batch extraction efficiencies for aqueous ammonium bicarbonate (42-50% total DU extracted), citric acid (30-42% total DU) and sulphuric acid (13-19% total DU) were evaluated. Characterisation of residues from bicarbonate-treated soils by synchrotron microfocus X-ray diffraction and X-ray absorption spectroscopy revealed partially leached U(IV)-oxide particles and some secondary uranyl-carbonate phases. Based on these data, a multi-stage extraction scheme was developed utilising leaching in ammonium bicarbonate followed by citric acid to dissolve secondary carbonate species. Site specific U extraction was improved to 68-87% total U by the application of this methodology, potentially providing a route to efficient DU decontamination using low cost, environmentally compatible reagents. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Phytotoxicity of trace metals in spiked and field-contaminated soils: Linking soil-extractable metals with toxicity.

    Science.gov (United States)

    Hamels, Fanny; Malevé, Jasmina; Sonnet, Philippe; Kleja, Dan Berggren; Smolders, Erik

    2014-11-01

    Soil tests have been widely developed to predict trace metal uptake by plants. The prediction of metal toxicity, however, has rarely been tested. The present study was set up to compare 8 established soil tests for diagnosing phytotoxicity in contaminated soils. Nine soils contaminated with Zn or Cu by metal mining, smelting, or processing were collected. Uncontaminated reference soils with similar soil properties were sampled, and series of increasing contamination were created by mixing each with the corresponding soil. In addition, each reference soil was spiked with either ZnCl2 or CuCl2 at several concentrations. Total metal toxicity to barley seedling growth in the field-contaminated soils was up to 30 times lower than that in corresponding spiked soils. Total metal (aqua regia-soluble) toxicity thresholds of 50% effective concentrations (EC50) varied by factors up to 260 (Zn) or 6 (Cu) among soils. For Zn, variations in EC50 thresholds decreased as aqua regia > 0.43 M HNO3  > 0.05 M ethylenediamine tetraacetic acid (EDTA) > 1 M NH4 NO3  > cobaltihexamine > diffusive gradients in thin films (DGT) > 0.001 M CaCl2 , suggesting that the last extraction is the most robust phytotoxicity index for Zn. The EDTA extraction was the most robust for Cu-contaminated soils. The isotopically exchangeable fraction of the total soil metal in the field-contaminated soils markedly explained the lower toxicity compared with spiked soils. The isotope exchange method can be used to translate soil metal limits derived from soils spiked with metal salts to site-specific soil metal limits. © 2014 SETAC.

  6. Evaluation and comparison of FTA card and CTAB DNA extraction methods for non-agricultural taxa 1

    OpenAIRE

    Siegel, Chloe S.; Stevenson, Florence O.; Zimmer, Elizabeth A.

    2017-01-01

    Premise of the study: An efficient, effective DNA extraction method is necessary for comprehensive analysis of plant genomes. This study analyzed the quality of DNA obtained using paper FTA cards prepared directly in the field when compared to the more traditional cetyltrimethylammonium bromide (CTAB)?based extraction methods from silica-dried samples. Methods: DNA was extracted using FTA cards according to the manufacturer?s protocol. In parallel, CTAB-based extractions were done using the a...

  7. DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.

    Science.gov (United States)

    Panova, Marina; Aronsson, Henrik; Cameron, R Andrew; Dahl, Peter; Godhe, Anna; Lind, Ulrika; Ortega-Martinez, Olga; Pereyra, Ricardo; Tesson, Sylvie V M; Wrange, Anna-Lisa; Blomberg, Anders; Johannesson, Kerstin

    2016-01-01

    The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths' different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects.

  8. Aqueous biphasic extraction of uranium and thorium from contaminated soils. Final report

    International Nuclear Information System (INIS)

    Chaiko, D.J.; Gartelmann, J.; Henriksen, J.L.; Krause, T.R.; Deepak; Vojta, Y.; Thuillet, E.; Mertz, C.J.

    1995-07-01

    The aqueous biphasic extraction (ABE) process for soil decontamination involves the selective partitioning of solutes and fine particulates between two immiscible aqueous phases. The biphase system is generated by the appropriate combination of a water-soluble polymer (e.g., polyethlene glycol) with an inorganic salt (e.g., sodium carbonate). Selective partitioning results in 99 to 99.5% of the soil being recovered in the cleaned-soil fraction, while only 0.5 to 1% is recovered in the contaminant concentrate. The ABE process is best suited to the recovery of ultrafine, refractory material from the silt and clay fractions of soils. During continuous countercurrent extraction tests with soil samples from the Fernald Environmental Management Project site (Fernald, OH), particulate thorium was extracted and concentrated between 6- and 16-fold, while the uranium concentration was reduced from about 500 mg/kg to about 77 mg/kg. Carbonate leaching alone was able to reduce the uranium concentration only to 146 mg/kg. Preliminary estimates for treatment costs are approximately $160 per ton of dry soil. A detailed flowsheet of the ABE process is provided

  9. Evaluation of methods for the extraction and purification of DNA from the human microbiome.

    Directory of Open Access Journals (Sweden)

    Sanqing Yuan

    Full Text Available DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled.In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used.Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.

  10. Extractability and bioavailability of Pb and As in historically contaminated orchard soil: Effects of compost amendments

    International Nuclear Information System (INIS)

    Fleming, Margaret; Tai, Yiping; Zhuang, Ping; McBride, Murray B.

    2013-01-01

    The availability of Pb and As in an historically contaminated orchard soil, after amendment with compost and aging in the field, was determined by single-step chemical extraction with 1.0 M ammonium acetate at pH 4.8, sequential extraction using the modified BCR test, and a redworm bioassay in the laboratory. The efficiency of soil Pb extraction by ammonium acetate was greater at higher total soil Pb but was reduced by compost amendment. Conversely, the extraction efficiency of total soil As increased with compost amendment, but was not sensitive to total soil As. The redworm bioassay indicated Pb (but not As) bioavailability to be reduced by soil amendment with compost, a result consistent with the ammonium acetate extraction test but not reflected in modified BCR test. Electron microprobe studies of the orchard soil revealed Pb and As to be spatially associated in discrete particles along with phosphorus and iron. -- Highlights: ► Soil Pb and As in an old orchard were concentrated in discrete particles. ► Compost amendment of contaminated soil reduced Pb bioavailability. ► Compost amendment of contaminated soil did not reduce As bioavailability. ► Ammonium acetate extraction test reflected bioavailability of soil Pb and As. -- Remediating metal-contaminated orchard soils with compost reduced lead bioavailability but had little effect on arsenic

  11. Impact of historical mining assessed in soils by kinetic extraction and lead isotopic ratios

    International Nuclear Information System (INIS)

    Camizuli, E.; Monna, F.; Bermond, A.; Manouchehri, N.; Besançon, S.; Losno, R.; Oort, F. van; Labanowski, J.; Perreira, A.; Chateau, C.; Alibert, P.

    2014-01-01

    The aim of this study is to estimate the long-term behaviour of trace metals, in two soils differently impacted by past mining. Topsoils from two 1 km 2 zones in the forested Morvan massif (France) were sampled to assess the spatial distribution of Cd, Cu, Pb and Zn. The first zone had been contaminated by historical mining. As expected, it exhibits higher trace-metal levels and greater spatial heterogeneity than the second non-contaminated zone, supposed to represent the local background. One soil profile from each zone was investigated in detail to estimate metal behaviour, and hence, bioavailability. Kinetic extractions were performed using EDTA on three samples: the A horizon from both soil profiles and the B horizon from the contaminated soil. For all three samples, kinetic extractions can be modelled by two first-order reactions. Similar kinetic behaviour was observed for all metals, but more metal was extracted from the contaminated A horizon than from the B horizon. More surprising is the general predominance of the residual fraction over the “labile” and “less labile” pools. Past anthropogenic inputs may have percolated over time through the soil profiles because of acidic pH conditions. Stable organo-metallic complexes may also have been formed over time, reducing metal availability. These processes are not mutually exclusive. After kinetic extraction, the lead isotopic compositions of the samples exhibited different signatures, related to contamination history and intrinsic soil parameters. However, no variation in lead signature was observed during the extraction experiment, demonstrating that the “labile” and “less labile” lead pools do not differ in terms of origin. Even if trace metals resulting from past mining and metallurgy persist in soils long after these activities have ceased, kinetic extractions suggest that metals, at least for these particular forest soils, do not represent a threat for biota. - Highlights: • Trace

  12. Impact of historical mining assessed in soils by kinetic extraction and lead isotopic ratios

    Energy Technology Data Exchange (ETDEWEB)

    Camizuli, E., E-mail: estelle.camizuli@u-bourgogne.fr [UMR 6298, ArTeHiS, Université de Bourgogne — CNRS — Culture, 6 bd Gabriel, Bat. Gabriel, 21000 Dijon (France); Monna, F. [UMR 6298, ArTeHiS, Université de Bourgogne — CNRS — Culture, 6 bd Gabriel, Bat. Gabriel, 21000 Dijon (France); Bermond, A.; Manouchehri, N.; Besançon, S. [Institut des sciences et industries du vivant et de l' environnement (AgroParisTech), Laboratoire de Chimie Analytique, 16, rue Claude Bernard, 75231 Paris Cedex 05 (France); Losno, R. [UMR 7583, LISA, Universités Paris 7-Paris 12 — CNRS, 61 av. du Gal de Gaulle, 94010 Créteil Cedex (France); Oort, F. van [UR 251, Pessac, Institut National de la Recherche Agronomique, Centre de Versailles-Grignon, RD 10, 78026 Versailles Cedex (France); Labanowski, J. [UMR 7285, IC2MP, Université de Poitiers — CNRS, 4, rue Michel Brunet, 86022 Poitiers (France); Perreira, A. [UMR 6298, ArTeHiS, Université de Bourgogne — CNRS — Culture, 6 bd Gabriel, Bat. Gabriel, 21000 Dijon (France); Chateau, C. [UFR SVTE, Université de Bourgogne, 6 bd Gabriel, Bat. Gabriel, 21000 Dijon (France); Alibert, P. [UMR 6282, Biogeosciences, Université de Bourgogne — CNRS, 6 bd Gabriel, Bat. Gabriel, 21000 Dijon (France)

    2014-02-01

    The aim of this study is to estimate the long-term behaviour of trace metals, in two soils differently impacted by past mining. Topsoils from two 1 km{sup 2} zones in the forested Morvan massif (France) were sampled to assess the spatial distribution of Cd, Cu, Pb and Zn. The first zone had been contaminated by historical mining. As expected, it exhibits higher trace-metal levels and greater spatial heterogeneity than the second non-contaminated zone, supposed to represent the local background. One soil profile from each zone was investigated in detail to estimate metal behaviour, and hence, bioavailability. Kinetic extractions were performed using EDTA on three samples: the A horizon from both soil profiles and the B horizon from the contaminated soil. For all three samples, kinetic extractions can be modelled by two first-order reactions. Similar kinetic behaviour was observed for all metals, but more metal was extracted from the contaminated A horizon than from the B horizon. More surprising is the general predominance of the residual fraction over the “labile” and “less labile” pools. Past anthropogenic inputs may have percolated over time through the soil profiles because of acidic pH conditions. Stable organo-metallic complexes may also have been formed over time, reducing metal availability. These processes are not mutually exclusive. After kinetic extraction, the lead isotopic compositions of the samples exhibited different signatures, related to contamination history and intrinsic soil parameters. However, no variation in lead signature was observed during the extraction experiment, demonstrating that the “labile” and “less labile” lead pools do not differ in terms of origin. Even if trace metals resulting from past mining and metallurgy persist in soils long after these activities have ceased, kinetic extractions suggest that metals, at least for these particular forest soils, do not represent a threat for biota. - Highlights: • Trace

  13. Extraction of heavy metals from contaminated soils using EDTA and HCl

    Directory of Open Access Journals (Sweden)

    Hatem Asel Gzar

    2015-01-01

    Full Text Available The present study examines the extraction of lead (Pb, cadmium (Cd and nickel (Ni from a contaminated soil by washing process. Ethylenediaminetetraacetic acid disodium salt (Na2EDTA and hydrochloric acid (HCl solution were used as extractants. Soil washing is one of the most suitable in-situ/ ex-situ remediation method in removing heavy metals. Soil was artificially contaminated with 500 mg/kg (Pb , Cd and Ni . A set of batch experiments were carried out at different conditions of extractant concentration , contact time, pH and agitation speed. The results showed that the maximum removal efficiencies of (Cd, Pb and Ni were (97, 88 and 24 % respectively using ( 0.1 M Na2EDTA. While the maximum removal efficiencies using (1M HCl were (98, 94 and 55% respectively. The experimental data of batch extraction were applied in four kinetic models; first order, parabolic diffusion, two constant and Elovich model. The parabolic diffusion was the most fitted to the experimental data.

  14. Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.

    Science.gov (United States)

    Ota, Hiroyuki; Lim, Tae-Kyu; Tanaka, Tsuyoshi; Yoshino, Tomoko; Harada, Manabu; Matsunaga, Tadashi

    2006-09-18

    A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods.

  15. Influence of Extractant and Soil Type on Molecular Characteristics of Humic Substances From Two Brazilian Soils

    Directory of Open Access Journals (Sweden)

    Dick Deborah Pinheiro

    1999-01-01

    Full Text Available In a previous study it was observed that humic substances (HS extracted with NaOH solution and with Na4P2O7 solution presented different molecular weights, and also that the extracted HS yield by each method varied between an Oxisol and a Mollisol from South Brazil. In the present study, we further investigated the organic matter in these soils by characterizing HS extracted with 0.5 mol L-1 NaOH and with neutral 0.15 mol L-1 Na4P2O7 solutions from the above mentioned samples, using elemental analysis and nuclear magnetic ressonance spectroscopy (liquid state ¹H- and 13C-NMR, and by relating the molecular differences to the extraction method and soil type. HS extracted with pyrophosphate were more humified, showing a higher aromaticity and higher carboxylic content. The NaOH-extracted HS were more aliphatic and contained a higher O-alkyl proportion, which is indicative of a less humified nature than the pyrophosphate-extracted HS.

  16. Improved Yield of High Molecular Weight DNA Coincides with Increased Microbial Diversity Access from Iron Oxide Cemented Sub-Surface Clay Environments

    Science.gov (United States)

    Hurt, Richard A.; Robeson, Michael S.; Shakya, Migun; Moberly, James G.; Vishnivetskaya, Tatiana A.; Gu, Baohua; Elias, Dwayne A.

    2014-01-01

    Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N2 grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1–V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered. PMID:25033199

  17. Targeting Unknowns Just Underfoot: Microbial Ecology and Community Genomics of C Cycling in Soil Informed and Enabled with DNA-SIP

    Science.gov (United States)

    Pepe-Ranney, C. P.; Campbell, A.; Buckley, D. H.

    2015-12-01

    Microorganisms drive biogeochemical cycles and because soil is a large global carbon (C) reservoir (soil contains more C than plants and the atmosphere combined), soil microorganisms are important players in the global C-cycle. Frustratingly, however, many soil microorganisms resist cultivation and soil communities are astoundingly complex. This makes soil microbiology difficult to study and without a solid understanding of soil microbial ecology, models of soil C feedbacks to climate change are under-informed. Stable isotope probing (SIP) is a useful approach for establishing identity-function connections in microbial communities but has been challenging to employ in soil due to the inadequate resolution of microbial community fingerprinting techniques. High throughput DNA sequencing improves SIP resolving power transforming it into a powerful tool for studying the soil C cycle. We conducted a DNA-SIP experiment to track flow of xylose-C, a labile component of plant biomass, and cellulose-C, the most abundant global biopolymer, through a soil microbial community. We could track 13C into microbial DNA even when added 13C amounted to less than 5% of native C and found Spartobacteria, Chloroflexi, and Planctomycetes taxa were among those that assimilated 13C cellulose. These lineages are cosmopolitan in soil but little is known of their ecophysiology. By profiling SSU rRNA genes across entire DNA-SIP density gradients, we assessed relative DNA atom % 13C per taxon in 13C treatments and found cellulose degraders exhibited signal consistent with a specialist lifestyle with respect to C preference. Further, DNA-SIP enriches DNA of targeted microorganisms (Verrucomicrobia cellulose degraders were enriched by nearly two orders of magnitude) and this enriched DNA can serve as template for community genomics. We produced draft genomes from soil cellulose degraders including microorganisms belonging to Verrucomicrobia, Chloroflexi, and Planctomycetes from SIP enriched DNA

  18. Evaluation and comparison of FTA card and CTAB DNA extraction methods for non-agricultural taxa.

    Science.gov (United States)

    Siegel, Chloe S; Stevenson, Florence O; Zimmer, Elizabeth A

    2017-02-01

    An efficient, effective DNA extraction method is necessary for comprehensive analysis of plant genomes. This study analyzed the quality of DNA obtained using paper FTA cards prepared directly in the field when compared to the more traditional cetyltrimethylammonium bromide (CTAB)-based extraction methods from silica-dried samples. DNA was extracted using FTA cards according to the manufacturer's protocol. In parallel, CTAB-based extractions were done using the automated AutoGen DNA isolation system. DNA quality for both methods was determined for 15 non-agricultural species collected in situ, by gel separation, spectrophotometry, fluorometry, and successful amplification and sequencing of nuclear and chloroplast gene markers. The FTA card extraction method yielded less concentrated, but also less fragmented samples than the CTAB-based technique. The card-extracted samples provided DNA that could be successfully amplified and sequenced. The FTA cards are also useful because the collected samples do not require refrigeration, extensive laboratory expertise, or as many hazardous chemicals as extractions using the CTAB-based technique. The relative success of the FTA card method in our study suggested that this method could be a valuable tool for studies in plant population genetics and conservation biology that may involve screening of hundreds of individual plants. The FTA cards, like the silica gel samples, do not contain plant material capable of propagation, and therefore do not require permits from the U.S. Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) for transportation.

  19. Minimally destructive DNA extraction from archaeological artefacts made from whale baleen

    DEFF Research Database (Denmark)

    Sinding, Mikkel Holger Strander; Gilbert, Tom; Grønnow, Bjarne

    2012-01-01

    Here we demonstrate the successful extraction and amplification of target species DNA from artefacts made of whale baleen collected from excavations of past palaeo-Eskimo and Inuit cultures in Greenland. DNA was successfully extracted and amplified from a single baleen bristle of 1.5 cm length...... genetic studies. We conclude that genetic investigation of historical baleen collections can contribute to our knowledge of the prehistoric population genetics of baleen whales, for example by quantifying the impact of modern whaling on the genetic diversity of bowhead whales....

  20. A two-stage extraction procedure for insensitive munition (IM) explosive compounds in soils.

    Science.gov (United States)

    Felt, Deborah; Gurtowski, Luke; Nestler, Catherine C; Johnson, Jared; Larson, Steven

    2016-12-01

    The Department of Defense (DoD) is developing a new category of insensitive munitions (IMs) that are more resistant to detonation or promulgation from external stimuli than traditional munition formulations. The new explosive constituent compounds are 2,4-dinitroanisole (DNAN), nitroguanidine (NQ), and nitrotriazolone (NTO). The production and use of IM formulations may result in interaction of IM component compounds with soil. The chemical properties of these IM compounds present unique challenges for extraction from environmental matrices such as soil. A two-stage extraction procedure was developed and tested using several soil types amended with known concentrations of IM compounds. This procedure incorporates both an acidified phase and an organic phase to account for the chemical properties of the IM compounds. The method detection limits (MDLs) for all IM compounds in all soil types were regulatory risk-based Regional Screening Level (RSL) criteria for soil proposed by the U.S. Army Public Health Center. At defined environmentally relevant concentrations, the average recovery of each IM compound in each soil type was consistent and greater than 85%. The two-stage extraction method decreased the influence of soil composition on IM compound recovery. UV analysis of NTO established an isosbestic point based on varied pH at a detection wavelength of 341 nm. The two-stage soil extraction method is equally effective for traditional munition compounds, a potentially important point when examining soils exposed to both traditional and insensitive munitions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Digital PCR for direct quantification of viruses without DNA extraction

    OpenAIRE

    Pav?i?, Jernej; ?el, Jana; Milavec, Mojca

    2015-01-01

    DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration mat...

  2. Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments

    International Nuclear Information System (INIS)

    Walia, S.; Khan, A.; Rosenthal, N.

    1990-01-01

    Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community

  3. Long-Term Effects of Multiwalled Carbon Nanotubes and Graphene on Microbial Communities in Dry Soil.

    Science.gov (United States)

    Ge, Yuan; Priester, John H; Mortimer, Monika; Chang, Chong Hyun; Ji, Zhaoxia; Schimel, Joshua P; Holden, Patricia A

    2016-04-05

    Little is known about the long-term effects of engineered carbonaceous nanomaterials (ECNMs) on soil microbial communities, especially when compared to possible effects of natural or industrial carbonaceous materials. To address these issues, we exposed dry grassland soil for 1 year to 1 mg g(-1) of either natural nanostructured material (biochar), industrial carbon black, three types of multiwalled carbon nanotubes (MWCNTs), or graphene. Soil microbial biomass was assessed by substrate induced respiration and by extractable DNA. Bacterial and fungal communities were examined by terminal restriction fragment length polymorphism (T-RFLP). Microbial activity was assessed by soil basal respiration. At day 0, there was no treatment effect on soil DNA or T-RFLP profiles, indicating negligible interference between the amended materials and the methods for DNA extraction, quantification, and community analysis. After a 1-year exposure, compared to the no amendment control, some treatments reduced soil DNA (e.g., biochar, all three MWCNT types, and graphene; P graphene); however, there were no significant differences across the amended treatments. These findings suggest that ECNMs may moderately affect dry soil microbial communities but that the effects are similar to those from natural and industrial carbonaceous materials, even after 1-year exposure.

  4. Mobility of radionuclides based on sequential extraction of soils

    International Nuclear Information System (INIS)

    Salbu, B.; Oughton, D.H.; Lien, H.N.; Oestby, G.; Strand, P.

    1992-01-01

    Since 1989, core samples of soil and vegetation from semi-natural pastures have been collected at selected sites in Norway during the growing season. The activity concentrations in soil and vegetation as well as transfer coefficients vary significantly between regions, within regions and even within sampling plot areas. In order to differentiate between mobil and inert fractions of radioactive and stable isotopes of Cs and Sr in soils, samples were extracted sequentially using agents with increasing dissolution power. The reproducibility of the sequential extraction technique is good and the data obtained seems most informative. As the distribution pattern for radioactive and stable isotopes of Cs and Sr are similar, a high degree of isotopic exchange is indicated. Based on easily leachable fractions, mobility factors are calculated. In general the mobility of 90 Sr is higher than for 137 Cs. Mobility factors are not significantly influenced by seasonal variations, but a decrease in the mobile fraction in soil with time is indicated. Mobility factors should be considered useful for modelling purposes. (au)

  5. Extraction of pesticides in soil using supercritical carbon dioxide co-solvents

    International Nuclear Information System (INIS)

    Forero, Jose R; Castro, Henry I; Guerrero, Jairo A.

    2009-01-01

    In this study, three organic solvents (ethyl acetate, methanol and acetone) were used as co solvent in supercritical fluid extraction (SFE) of a mixture of pesticides with different physical and chemical properties present in soil. These pesticides were determined by gas chromatography with electronic micro capture detector μECD and nitrogen-phosphorus detector (NPD), coupled in parallel. The extractions were performed on spiked soil samples using supercritical carbon dioxide (CO 2 SC) as the extracting phase to 35 celsius degrade and 14 MPa, using 10 mL of each co solvent and it was found that methanol offers the greatest efficiency in the extraction process obtaining recovery values between 51.24 and 123.50%.

  6. Influence of soil-extractable aluminium and pH on the uptake of aluminium from soil into the soybean plant (Glycine max).

    Science.gov (United States)

    Dong, D; Thornton, I; Ramsey, M H

    1993-09-01

    The effects of soil pH and other soil properties on the uptake of AI by soybean plants have been investigated in a greenhouse experiment. Six soils were compared that were developed over six contrasting bedrock types ranging widely in their AI content and other chemical and physical characteristics, namely Oxford Clay, Chalk, Lower Lias Clay, Devonian Shale, Granite and Lower Greensand. Soil pH varied naturally between soil types and each soil was also amended to give two other pH levels using elemental sulphur and/or calcium carbonate. AI concentrations in various parts of the soybean plants were determined by ICP-AES after acid digestion. The AI solubility in the soils and hence its availability to the plants was estimated using a number of different reagents designed to extract different forms of AI.The AI concentration measured in the soybean leaves was found to be predicted most accurately by the 'available' AI extracted from soils by 0.02 M CaCl2. The relationship appears to the linear, with a correlation coefficient of 0.97 (p <0.01). The AI content of the leaves increases with decreasing soil pH. The relationship is non-linear with a marked increase in leaf AI for soils with pH <4.4. The amounts of 'plant-available' AI in the soils extracted with 0.02 M CaCl2 was much less than that extracted with 0.05 M EDTA, although both increased markedly with decreasing soil pH. The amount of AI measured in the soybean plants was directly related to both the 'available' forms of AI in the soils, and also to the pH of the soils. Soil pH was identified as a major factor that controls the uptake of Al from soil into the soybean plant.

  7. Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels.

    Science.gov (United States)

    Fredlund, Elisabeth; Gidlund, Ann; Olsen, Monica; Börjesson, Thomas; Spliid, Niels Henrik Hytte; Simonsson, Magnus

    2008-04-01

    Identification of Fusarium species by traditional methods requires specific skill and experience and there is an increased interest for new molecular methods for identification and quantification of Fusarium from food and feed samples. Real-time PCR with probe technology (Taqman) can be used for the identification and quantification of several species of Fusarium from cereal grain samples. There are several critical steps that need to be considered when establishing a real-time PCR-based method for DNA quantification, including extraction of DNA from the samples. In this study, several DNA extraction methods were evaluated, including the DNeasy Plant Mini Spin Columns (Qiagen), the Bio robot EZ1 (Qiagen) with the DNeasy Blood and Tissue Kit (Qiagen), and the Fast-DNA Spin Kit for Soil (Qbiogene). Parameters such as DNA quality and stability, PCR inhibitors, and PCR efficiency were investigated. Our results showed that all methods gave good PCR efficiency (above 90%) and DNA stability whereas the DNeasy Plant Mini Spin Columns in combination with sonication gave the best results with respect to Fusarium DNA yield. The modified DNeasy Plant Mini Spin protocol was used to analyse 31 wheat samples for the presence of F. graminearum and F. culmorum. The DNA level of F. graminearum could be correlated to the level of DON (r(2) = 0.9) and ZEN (r(2) = 0.6) whereas no correlation was found between F. culmorum and DON/ZEA. This shows that F. graminearum and not F. culmorum, was the main producer of DON in Swedish wheat during 2006.

  8. Use of extracts from oyster shell and soil for cultivation of Spirulina maxima.

    Science.gov (United States)

    Jung, Joo-Young; Kim, Sunmin; Lee, Hansol; Kim, Kyochan; Kim, Woong; Park, Min S; Kwon, Jong-Hee; Yang, Ji-Won

    2014-12-01

    Calcium ion and trace metals play important roles in various metabolisms of photosynthetic organisms. In this study, simple methods were developed to extract calcium ion and micronutrients from oyster shell and common soil, and the prepared extracts were tested as a replacement of the corresponding chemicals that are essential for growth of microalgae. The oyster shell and soil were treated with 0.1 M sodium hydroxide or with 10 % hydrogen peroxide, respectively. The potential application of these natural sources to cultivation was investigated with Spirulina maxima. When compared to standard Zarrouk medium, the Spirulina maxima cultivated in a modified Zarrouk media with elements from oyster shell and soil extract exhibited increases in biomass, chlorophyll, and phycocyanin by 17, 16, and 64 %, respectively. These results indicate that the extracts of oyster shell and soil provide sufficient amounts of calcium and trace metals for successful cultivation of Spirulina maxima.

  9. Noninvasive genetic sampling of endangered muriqui (Primates, Atelidae: efficiency of fecal DNA extraction

    Directory of Open Access Journals (Sweden)

    Paulo B. Chaves

    2006-01-01

    Full Text Available The muriqui (Brachyteles is one of the most endangered primates in the world, however little is known about the viability of the remaining populations. We evaluated the technique of extracting DNA from wild muriqui feces for PCR applications. In order to determine the effect of the DNA in subsequent amplifications, we analyzed five different extracts. The importance of the recommended BSA and the HotStarTaq DNA polymerase was tested. The minimal conditions to successfully amplify highly degraded fecal DNA were determined, showing that the recommended reagents are not required. We envision that this method may be useful in further conservation management studies.

  10. Designing easy DNA extraction: Teaching creativity through laboratory practice.

    Science.gov (United States)

    Susantini, Endang; Lisdiana, Lisa; Isnawati; Tanzih Al Haq, Aushia; Trimulyono, Guntur

    2017-05-01

    Subject material concerning Deoxyribose Nucleic Acid (DNA) structure in the format of creativity-driven laboratory practice offers meaningful learning experience to the students. Therefore, a laboratory practice in which utilizes simple procedures and easy-safe-affordable household materials should be promoted to students to develop their creativity. This study aimed to examine whether designing and conducting DNA extraction with household materials could foster students' creative thinking. We also described how this laboratory practice affected students' knowledge and views. A total of 47 students participated in this study. These students were grouped and asked to utilize available household materials and modify procedures using hands-on worksheet. Result showed that this approach encouraged creative thinking as well as improved subject-related knowledge. Students also demonstrated positive views about content knowledge, social skills, and creative thinking skills. This study implies that extracting DNA with household materials is able to develop content knowledge, social skills, and creative thinking of the students. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(3):216-225, 2017. © 2016 The International Union of Biochemistry and Molecular Biology.

  11. Polyaspartate extraction of cadmium ions from contaminated soil: Evaluation and optimization using central composite design.

    Science.gov (United States)

    Mu'azu, Nuhu Dalhat; Haladu, Shamsuddeen A; Jarrah, Nabeel; Zubair, Mukarram; Essa, Mohammad H; Ali, Shaikh A

    2018-01-15

    The occurrences of heavy metal contaminated sites and soils and the need for devising environmentally friendly solutions have become global issues of serious concern. In this study, polyaspartate (a highly biodegradable agent) was synthesized using L-Aspartic acid via a new modified thermal procedure and employed for extraction of cadmium ions (Cd) from contaminated soil. Response surface methodology approach using 3 5 full faced centered central composite design was employed for modeling, evaluating and optimizing the influence of polyaspartate concentration (36-145mM), polyaspartate/soil ratio (5-25), initial heavy metal concentration (100-500mg/kg), initial pH (3-6) and extraction time (6-24h) on Cd ions extracted into the polyaspartate solution and its residual concentration in the treated soil. The Cd extraction efficacy obtained reached up to 98.8%. Increase in Cd extraction efficiency was associated with increase in the polyaspartate and Cd concentration coupled with lower polyaspertate/soil ratio and initial pH. Under the optimal conditions characterized with minimal utilization of the polyaspartate and high Cd ions removal, the extractible Cd in the polyaspartate solution reached up to 84.4mg/L which yielded 85% Cd extraction efficacy. This study demonstrates the suitability of using polyaspartate as an effective environmentally friendly chelating agent for Cd extraction from contaminated soils. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. 1,4-Dioxane Remediation by Extreme Soil Vapor Extraction (XSVE). Screening-Level Feasibility Assessment and Design Tool in Support of 1,4-Dioxane Remediation by Extreme Soil Vapor Extraction (XSVE) ESTCP Project ER 201326

    Science.gov (United States)

    2017-10-01

    USER GUIDE 1,4-Dioxane Remediation by Extreme Soil Vapor Extraction (XSVE) Screening-Level Feasibility Assessment and Design Tool in...Support of 1,4-Dioxane Remediation by Extreme Soil Vapor Extraction (XSVE) ESTCP Project ER-201326 OCTOBER 2017 Rob Hinchee Integrated Science...Technology, Inc. 1509 Coastal Highway Panacea, FL 32346 8/8/2013 - 8/8/2018 10-2017 1,4-Dioxane Remediation by Extreme Soil Vapor Extraction (XSVE) Screening

  13. Simple DNA extraction of urine samples: Effects of storage temperature and storage time.

    Science.gov (United States)

    Ng, Huey Hian; Ang, Hwee Chen; Hoe, See Ying; Lim, Mae-Lynn; Tai, Hua Eng; Soh, Richard Choon Hock; Syn, Christopher Kiu-Choong

    2018-06-01

    Urine samples are commonly analysed in cases with suspected illicit drug consumption. In events of alleged sample mishandling, urine sample source identification may be necessary. A simple DNA extraction procedure suitable for STR typing of urine samples was established on the Promega Maxwell ® 16 paramagnetic silica bead platform. A small sample volume of 1.7mL was used. Samples were stored at room temperature, 4°C and -20°C for 100days to investigate the influence of storage temperature and time on extracted DNA quantity and success rate of STR typing. Samples stored at room temperature exhibited a faster decline in DNA yield with time and lower typing success rates as compared to those at 4°C and -20°C. This trend can likely be attributed to DNA degradation. In conclusion, this study presents a quick and effective DNA extraction protocol from a small urine volume stored for up to 100days at 4°C and -20°C. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Quantification of Plasmodiophora brassicae Using a DNA-Based Soil Test Facilitates Sustainable Oilseed Rape Production

    OpenAIRE

    Ann-Charlotte Wallenhammar; Albin Gunnarson; Fredrik Hansson; Anders Jonsson

    2016-01-01

    Outbreaks of clubroot disease caused by the soil-borne obligate parasite Plasmodiophora brassicae are common in oilseed rape (OSR) in Sweden. A DNA-based soil testing service that identifies fields where P. brassicae poses a significant risk of clubroot infection is now commercially available. It was applied here in field surveys to monitor the prevalence of P. brassicae DNA in field soils intended for winter OSR production and winter OSR field experiments. In 2013 in Scania, prior to plantin...

  15. Factors influencing the chemical extractability of 241Am from a contaminated soil

    International Nuclear Information System (INIS)

    Nishita, H.; Hamilton, M.

    1976-01-01

    Factors influencing the extractability of 241 Am from an artificially contaminated soil were investigated. This was done with an equilibrium batch technique using CH 3 COOH-NH 4 OH and HNO 3 -NaOH extracting systems. The influence of several soil components was determined indirectly by selectively removing them from the soil. The effect of water- and HCl-soluble salts and organic matter on 241 Am extractability was small. The most marked effect was due to the soil organic fraction that was not water- or HCl-soluble. This organic fraction was influential under both low and high pH conditions, but its influence was particularly marked under low pH conditions. The free iron-oxides had an appreciable effect under low pH conditions, but no observable effect in the high pH range. Though to a lesser extent, the free silica and alumina, amorphous alumino-silicate, and possibly residual organic matter also showed some influence. These results provide some implications on the conditions that influence the movement of 241 Am in soils and its availability to plants. A review of the literature on the behavior of Am in soils is included

  16. Prediction of reducible soil iron content from iron extraction data

    NARCIS (Netherlands)

    Bodegom, van P.M.; Reeven, van J.; Denier van der Gon, H.A.C.

    2003-01-01

    Soils contain various iron compounds that differ in solubility, reducibility and extractability. Moreover, the contribution of the various iron compounds to total iron (Fe) and total Fe concentrations differs highly among soils. As a result, the total reducible Fe content can also differ among

  17. Influence of soil and hydrocarbon properties on the solvent extraction of high-concentration weathered petroleum from contaminated soils.

    Science.gov (United States)

    Sui, Hong; Hua, Zhengtao; Li, Xingang; Li, Hong; Wu, Guozhong

    2014-05-01

    Petroleum ether was used to extract petroleum hydrocarbons from soils collected from six oil fields with different history of exploratory and contamination. It was capable of fast removing 76-94 % of the total petroleum hydrocarbons including 25 alkanes (C11-C35) and 16 US EPA priority polycyclic aromatic hydrocarbons from soils at room temperature. The partial least squares analysis indicated that the solvent extraction efficiencies were positively correlated with soil organic matter, cation exchange capacity, moisture, pH, and sand content of soils, while negative effects were observed in the properties reflecting the molecular size (e.g., molecular weight and number of carbon atoms) and hydrophobicity (e.g., water solubility, octanol-water partition coefficient, soil organic carbon partition coefficient) of hydrocarbons. The high concentration of weathered crude oil at the order of 10(5) mg kg(-1) in this study was demonstrated adverse for solvent extraction by providing an obvious nonaqueous phase liquid phase for hydrocarbon sinking and increasing the sequestration of soluble hydrocarbons in the insoluble oil fractions during weathering. A full picture of the mass distribution and transport mechanism of petroleum contaminants in soils will ultimately require a variety of studies to gain insights into the dynamic interactions between environmental indicator hydrocarbons and their host oil matrix.

  18. A multistratum approach to soil vapor extraction

    International Nuclear Information System (INIS)

    Fuhr, J.M.; Giesler, R.S.

    1993-01-01

    An innovative soil remediation design was implemented to address petroleum hydrocarbon contamination in a gradationally stratified subsurface environment containing alternating layers of clay, sand and clayey sand, and perched water tables in north Florida. The soil vapor extraction (SVE) design enables remediation to focus on distinct subsurface intervals depending on changing site conditions such as constituent concentration levels and periodic water-table fluctuations. Contaminated soils were assessed from the land surface to the top of a two foot thick perched water table located at 13 feet below land surface (bls), and also were encountered below the perched water table downward to another perched water table at 45 feet bls. Use of an organic vapor analyzer equipped with a flame ionization detector revealed hydrocarbon vapor concentrations in soil samples ranging to greater than 1,000 parts per million (ppm). Nonaqueous phase liquids were encountered on both perched water tables. Based on the site assessment, a multistratum soil and ground-water remediation system was designed and constructed. A pilot test was conducted to aid in the design of an effective SVE system

  19. Identifikasi bite marks dengan ekstraksi DNA metode Chelex (Bite marks identification with Chelex methods in DNA extraction

    Directory of Open Access Journals (Sweden)

    Imelda Kristina Sutrisno

    2013-06-01

    Full Text Available Background: In the case of crime often encountered evidence in bite marks form that was found on the victim’s body. Generally, bitemarks identification use standard techniques that compare the interpretation picture with the tooth model of suspected person. However, sometimes the techniques do not obtain accurate results. Therefore another technique is needed to support the identification process,such as DNA analysis that use the remaining epithelium attached in saliva to identify the DNA of the suspected person. In this processes a limited DNA material could be met, not only less in quantity but also less in quality. Chelex known as one of an effective DNA extraction method in DNA forensic case is needed to overcome this problem. Purpose: The study was aimed to examine the use of Chelex as DNA extraction method on a bitemarks sample models. Methods: The blood and bitemarks of 5 persons with were taken. The DNA of each subject was exctracted with Chelex and quantified the quantity with UV Spechtrophotometer. The DNA results was amplified by PCR at locus vWA and TH01 then vizualised by electrophoresis. Results: The electrophoresis’s results showed band at locus vWA and TH01 for blood sample and bite marks with no significant differences. Conclusion: The study showed that Chelex method could be use to extract DNA from bitemarks.Latar belakang: Dalam kasus kejahatan sering dijumpai bukti dalam bentuk bekas gigitan (bitemarks yang ditemukan pada tubuh korban. Umumnya, untuk mengidentifikasi bite marks menggunakan teknik standar yaitu membandingkan foto interpretasi dengan model gigi dari orang yang dicurigai. Namun demikian teknik ini terkadang tidak mendapatkan hasil yang akurat, sehingga diperlukan teknik lain untuk menunjang keberhasilan proses identifikasi pelaku, yakni melalui analisis DNA bitemarks, yang diperoleh dari saliva yang mengandung sisa epitel tersangka pelaku. Sampel DNA yang berasal dari bitemarks umumnya terbatas, tidak hanya

  20. DNA in ancient bone - where is it located and how should we extract it?

    DEFF Research Database (Denmark)

    Campos, Paula; Craig, Oliver E.; Turner-Walker, Gordon

    2012-01-01

    Despite the widespread use of bones in ancient DNA (aDNA) studies, relatively little concrete information exists in regard to how the DNA in mineralised collagen degrades, or where it survives in the material's architecture. While, at the macrostructural level, physical exclusion of microbes...... and other external contaminants may be an important feature, and, at the ultrastructural level, the adsorption of DNA to hydroxyapatite and/or binding of DNA to Type I collagen may stabilise the DNA, the relative contribution of each, and what other factors may be relevant, are unclear....... The question arises as to whether this may be due to post-collection preservation or just an artefact of the extraction methods used in these different studies? In an attempt to resolve these questions, we examine the efficacy of DNA extraction methods, and the quality and quantity of DNA recovered from both...

  1. Determination of diagnostic standards on saturated soil extracts for cut roses grown in greenhouses.

    Science.gov (United States)

    Franco-Hermida, John Jairo; Quintero, María Fernanda; Cabrera, Raúl Iskander; Guzman, José Miguel

    2017-01-01

    This work comprises the theoretical determination and validation of diagnostic standards for the analysis of saturated soil extracts for cut rose flower crops (Rosa spp.) growing in the Bogota Plateau, Colombia. The data included 684 plant tissue analyses and 684 corresponding analyses of saturated soil extracts, all collected between January 2009 and June 2013. The tissue and soil samples were selected from 13 rose farms, and from cultivars grafted on the 'Natal Briar' rootstock. These concurrent samples of soil and plant tissues represented 251 production units (locations) of approximately 10,000 m2 distributed across the study area. The standards were conceived as a tool to improve the nutritional balance in the leaf tissue of rose plants and thereby define the norms for expressing optimum productive potential relative to nutritional conditions in the soil. To this end, previously determined diagnostic standard for rose leaf tissues were employed to obtain rates of foliar nutritional balance at each analyzed location and as criteria for determining the diagnostic norms for saturated soil extracts. Implementing this methodology to foliar analysis, showed a higher significant correlation for diagnostic indices. A similar behavior was observed in saturated soil extracts analysis, becoming a powerful tool for integrated nutritional diagnosis. Leaf analyses determine the most limiting nutrients for high yield and analyses of saturated soil extracts facilitate the possibility of correcting the fertigation formulations applied to soils or substrates. Recommendations are proposed to improve the balance in soil-plant system with which the possibility of yield increase becomes more probable. The main recommendations to increase and improve rose crop flower yields would be: continuously check pH values of SSE, reduce the amounts of P, Fe, Zn and Cu in fertigation solutions and carefully analyze the situation of Mn in the soil-plant system.

  2. Variability of standard artificial soils: Physico-chemical properties and phenanthrene desorption measured by means of supercritical fluid extraction

    International Nuclear Information System (INIS)

    Bielská, Lucie; Hovorková, Ivana; Komprdová, Klára; Hofman, Jakub

    2012-01-01

    The study is focused on artificial soil which is supposed to be a standardized “soil like” medium. We compared physico-chemical properties and extractability of Phenanthrene from 25 artificial soils prepared according to OECD standardized procedures at different laboratories. A substantial range of soil properties was found, also for parameters which should be standardized because they have an important influence on the bioavailability of pollutants (e.g. total organic carbon ranged from 1.4 to 6.1%). The extractability of Phe was measured by supercritical fluid extraction (SFE) at harsh and mild conditions. Highly variable Phe extractability from different soils (3–89%) was observed. The extractability was strongly related (R 2 = 0.87) to total organic carbon content, 0.1–2 mm particle size, and humic/fulvic acid ratio in the following multiple regression model: SFE (%) = 1.35 * sand (%) − 0.77 * TOC (%)2 + 0.27 * HA/FA. - Highlights: ► We compared properties and extractability of Phe from 25 different artificial soils. ► Substantial range of soil properties was found, also for important parameters. ► Phe extractability was measured by supercritical fluid extraction (SFE) at 2 modes. ► Phe extractability was highly variable from different soils (3–89%). ► Extractability was strongly related to TOC, 0.1–2 mm particles, and HA/FA. - Significant variability in physico-chemical properties exists between artificial soils prepared at different laboratories and affects behavior of contaminants in these soils.

  3. Development of an efficient fungal DNA extraction method to be used in random amplified polymorphic DNA-PCR analysis to differentiate cyclopiazonic acid mold producers.

    Science.gov (United States)

    Sánchez, Beatriz; Rodríguez, Mar; Casado, Eva M; Martín, Alberto; Córdoba, Juan J

    2008-12-01

    A variety of previously established mechanical and chemical treatments to achieve fungal cell lysis combined with a semiautomatic system operated by a vacuum pump were tested to obtain DNA extract to be directly used in randomly amplified polymorphic DNA (RAPD)-PCR to differentiate cyclopiazonic acid-producing and -nonproducing mold strains. A DNA extraction method that includes digestion with proteinase K and lyticase prior to using a mortar and pestle grinding and a semiautomatic vacuum system yielded DNA of high quality in all the fungal strains and species tested, at concentrations ranging from 17 to 89 ng/microl in 150 microl of the final DNA extract. Two microliters of DNA extracted with this method was directly used for RAPD-PCR using primer (GACA)4. Reproducible RAPD fingerprints showing high differences between producer and nonproducer strains were observed. These differences in the RAPD patterns did not differentiate all the strains tested in clusters by cyclopiazonic acid production but may be very useful to distinguish cyclopiazonic acid producer strains from nonproducer strains by a simple RAPD analysis. Thus, the DNA extracts obtained could be used directly without previous purification and quantification for RAPD analysis to differentiate cyclopiazonic acid producer from nonproducer mold strains. This combined analysis could be adaptable to other toxigenic fungal species to enable differentiation of toxigenic and non-toxigenic molds, a procedure of great interest in food safety.

  4. Effect of gamma-irradiation on rice seed DNA. Pt. 1. Yield and molecular size of DNA extracted from irradiated rice seeds

    International Nuclear Information System (INIS)

    Kawamura, Yoko; Konishi, Akihiro; Yamada, Takashi; Saito, Yukio

    1995-01-01

    The effect of gamma-irradiation on the DNA of hulled rice seeds was investigated. The cetyltrimethylammonium bromide (CTAB) method was preferred for the extraction of DNA from rice seeds because of its high quality and good yield. The yield of DNA that was determined by gel electrophoresis, decreased as the irradiation dose increased from 1 kGy. DNA extracted from rice seeds irradiated with a 30 kGy dose showed a molecular size of less than 20 kb, while that from unirradiated rice showed more than 100 kb in electrophoretic profiles. It can be assumed that the decrease in yield was mainly induced by the crosslinking between protein and DNA, and the reduction in molecular size was induced by double-strand breaks. (J.P.N.)

  5. Cadmium and zinc in soil solution extracts following the application of phosphate fertilizers.

    Science.gov (United States)

    Lambert, Raphaël; Grant, Cynthia; Sauvé, Sébastien

    2007-06-01

    This study investigated the solubility of cadmium and zinc in soils after the application of phosphate fertilizers containing those two metals. The solubility of cadmium and zinc was assessed by measuring their concentration in soil water extracts. Three monoammonium phosphate fertilizers containing various amounts of metals were applied on cultivated fields for 3 years at three different rates. In order to investigate the effects of long-term applications of fertilizers on the solubility of Cd and Zn, a similar design was used to apply contaminated fertilizers to soils in a laboratory experiment using a single fertilizer addition equivalent to 15 years of application. Phosphate fertilizers increased the concentration of Cd in soil extracts compared to control in 87% and 80% of the treatments in field and laboratory experiments respectively. Both increasing the rate of application and using fertilizer containing more Cd lead to higher Cd concentrations in extracts for the field and the laboratory experiments. The addition of the equivalent of 15 years of fertilizer application in the laboratory results in higher Cd concentration in extracts compared to the field experiment. For Zn, the fertilizer treatments enhanced the metal solution concentration in 83% of field treatments, but no significant correlations could be found between Zn inputs and its concentration in solution. In the laboratory, fertilizer additions increase the Zn concentrations in 53% of the treatments and decrease it in most of the other treatments. The decrease in Zn concentrations in the laboratory trial is attributed to the higher phosphate concentrations in the soil solution; which is presumed to have contributed to the precipitation of Zn-phosphates. For both trials, the metal concentrations in soil extracts cannot be related to the Zn concentration in the fertilizer or the rate of application. The high Zn to Cd ratio is presumably responsible for the Cd increase in the soil extracts due to

  6. Occurrence of pesticide non extractable residues in physical and chemical fractions from two natural soils.

    Science.gov (United States)

    Andreou, K.; Jones, K.; Semple, K.

    2009-04-01

    Distribution of pesticide non extractable residues resulted from the incubation of two natural soils with each of the isoproturon, diazinon and cypermethrin pesticide was assessed in this study. Pesticide non extractable residues distribution in soil physical and chemical fractions is known to ultimately affect their fate. This study aimed to address the fate and behaviour of the non extractable residues in the context of their association with soil physical and chemical fractions with varying properties and characteristics. Non extractable residues were formed from incubation of each pesticide in the two natural soils over a period of 24 months. Soils containing the non extractable residues were fractionated into three solid phase fractions using a physical fractionation procedure as follows: Sediment (SED, >20 μm), (II) Microaggregate (MA, 20-2 μm) and (III) Colloid phase (COL, 2-0.05 μm). Each soil fraction was then fractionated into organic carbon chemical fractionations as follows: Fulvic acid (FA), Humic acid (HA) and Humin (HM). Significant amount of the pesticides was lost during the incubation period. Enrichment factors for the organic carbon and the 14C-pesticide residues were higher in the MA and COL fraction rather than the SED fraction. Greater association and enrichment of the fulvic acid fraction of the organic carbon in the soil was observed. Non extractable residues at the FA fraction showed to diminish while in the HA fraction were increased with decreasing the fraction size. An appreciable amount of non extractable residues were located in the HM fraction but this was less than the amount recovered in the humic substances. Long term fate of pesticide non extractable residues in the soil structural components is important in order to assess any risk associated with them.

  7. Comparison of nine DNA extraction methods for the diagnosis of bovine tuberculosis by real time PCR

    OpenAIRE

    Moura, André; Hodon, Mikael Arrais; Soares Filho, Paulo Martins; Issa, Marina de Azevedo; Oliveira, Ana Paula Ferreira de; Fonseca Júnior, Antônio Augusto

    2016-01-01

    ABSTRACT: Bovine tuberculosis is an infectious disease with a high impact on the cattle industry, particularly in developing countries. PCR is a very sensitive method for detection of infectious agents, but the sensitivity of molecular diagnosis is largely dependent on the efficiency of the DNA extraction methods. The objective of this study was to evaluate DNA extraction methods for direct detection of Mycobacterium bovis in bovine tissue. Nine commercial kits for DNA extraction were evalua...

  8. Extraction of an urease-active organo-complex from soil.

    Science.gov (United States)

    Burns, R. G.; El-Sayed, M. H.; Mclaren, A. D.

    1972-01-01

    Description of an extraction from a Dublin clay loam soil of a colloidal organic matter complex that is urease active and, by X-ray analysis, free of clays. Urease activity in the clay-free precipitates, as in the soil, was not destroyed by the activity of an added proteolytic enzyme, pronase. This is attributed to the circumstance that native soil urease resides in organic colloidal particles with pores large enough for water, urea, ammonia, and carbon dioxide to pass freely, but nevertheless small enough to exclude pronase.

  9. Simplified extraction of good quality genomic DNA from a variety of ...

    African Journals Online (AJOL)

    Depending on the nature and complexity of plant material, proper method needs to be employed for extraction of genomic DNA, along with its performance evaluation by different molecular techniques. Here, we optimized and employed a simple genomic DNA isolation protocol suitable for a variety of plant materials ...

  10. Resistive heating enhanced soil vapor extraction of chlorinated solvents from trichloroethylene contaminated silty, low permeable soil

    NARCIS (Netherlands)

    Zutphen, M. van; Heron, G.; Enfield, C.G.; Christensen, T.H.

    1998-01-01

    A 2D-laboratory box experiment (12 x 56 x 116 cm) was conducted to simulate the enhancement of soil vapor extraction by the application of low frequency electrical heating Uoule heating) for the remediation of a low permeable, silty soil contaminated with trichloroethylene. Joule heating enlarged

  11. Selective Extraction of Organic Contaminants from Soil Using Pressurised Liquid Extraction

    Directory of Open Access Journals (Sweden)

    Rozita Osman

    2013-01-01

    Full Text Available This study focuses on the application of sorbents in pressurised liquid extraction (PLE cell to establish a selective extraction of a variety of organic contaminants (polycyclic aromatic hydrocarbons (PAHs, chlorpyrifos, phenol, pentachlorophenol, and sterols from soil. The selectivity and efficiency of each sorbent depend on the properties of the material, extracting solvent, capacity factor, organic compounds of interest, and PLE operating parameters (temperature, pressure, and extraction time. Several sorbents (silica, alumina, and Florisil were evaluated and with the proper choice of solvents, polar and nonpolar compounds were successfully separated in two fractions. Nonpolar compounds (PAHs, chlorpyrifos, and pentachlorophenol were recovered in the first fraction using a polar sorbent such as Florisil or alumina, and n-hexane as eluting solvent, while more polar compounds (phenol and sterols were recovered in the second fraction using methanol. Silica (5 g was found to be effective for selective extraction with the satisfactory recoveries for all compounds (PAHs from 87.1–96.2%, chlorpyrifos 102.9%, sterols from 93.7–100.5%, phenol 91.9%, and pentachlorophenol 106.2%. The efficiency and precision of this extraction approach and the existing EPA Method 3545 were compared.

  12. Selenium speciation in phosphate mine soils and evaluation of a sequential extraction procedure using XAFS

    International Nuclear Information System (INIS)

    Favorito, Jessica E.; Luxton, Todd P.; Eick, Matthew J.; Grossl, Paul R.

    2017-01-01

    Selenium is a trace element found in western US soils, where ingestion of Se-accumulating plants has resulted in livestock fatalities. Therefore, a reliable understanding of Se speciation and bioavailability is critical for effective mitigation. Sequential extraction procedures (SEP) are often employed to examine Se phases and speciation in contaminated soils but may be limited by experimental conditions. We examined the validity of a SEP using X-ray absorption spectroscopy (XAS) for both whole and a sequence of extracted soils. The sequence included removal of soluble, PO 4 -extractable, carbonate, amorphous Fe-oxide, crystalline Fe-oxide, organic, and residual Se forms. For whole soils, XANES analyses indicated Se(0) and Se(-II) predominated, with lower amounts of Se(IV) present, related to carbonates and Fe-oxides. Oxidized Se species were more elevated and residual/elemental Se was lower than previous SEP results from ICP-AES suggested. For soils from the SEP sequence, XANES results indicated only partial recovery of carbonate, Fe-oxide and organic Se. This suggests Se was incompletely removed during designated extractions, possibly due to lack of mineral solubilization or reagent specificity. Selenium fractions associated with Fe-oxides were reduced in amount or removed after using hydroxylamine HCl for most soils examined. XANES results indicate partial dissolution of solid-phases may occur during extraction processes. This study demonstrates why precautions should be taken to improve the validity of SEPs. Mineralogical and chemical characterizations should be completed prior to SEP implementation to identify extractable phases or mineral components that may influence extraction effectiveness. Sequential extraction procedures can be appropriately tailored for reliable quantification of speciation in contaminated soils. - Highlights: • XANES spectra indicated whole soils consisted of mostly elemental and organic Se and lower amounts of sorbed oxidized Se.

  13. An inexpensive and rapid method for extracting papilionoid genomic DNA from herbarium specimens.

    Science.gov (United States)

    Riahi, M; Zarre, S; Maassoumi, A A; Attar, F; Kazempour Osaloo, S

    2010-07-13

    Three DNA extraction protocols were compared for their ability to yield DNA from the leaves of herbarium specimens of nine species from nine genera of the Papilionoideae. We tested two protocols that use classic procedures for lysis and purification with cetyl trimethylammonium bromide (CTAB); a third protocol used a Nucleospin Plant kit. DNA obtained from all three procedures was quantified and tested by PCR. Test results indicated the superiority of one of the CTAB protocols. We made some modifications, developing a protocol that produced high-quality DNA from all nine species. The modification involved the use of a lower EDTA concentration (20 mM instead of 50 mM) and a higher beta-mercaptoethanol concentration (1% instead of 0.4%) in the extraction buffer. The modified protocol avoids the necessity for a second DNA precipitation step. This new CTAB protocol includes the use of 1.4 M NaCl, 20 mM EDTA and 1% beta-mercaptoethanol in the extraction; DNA precipitation time is reduced. A reduction in contaminating metabolites (such as PCR inhibitors) in the sample mixtures and lower costs for reagents are characteristics of this modified protocol; the cost of analysis per sample was lowered, compared to previous options. The quality of DNA was suitable for PCR amplification. This is a practical alternative to more difficult, time-consuming and expensive protocols.

  14. Optimization of microwave-assisted extraction and supercritical fluid extraction of carbamate pesticides in soil by experimental design methodology.

    Science.gov (United States)

    Sun, Lei; Lee, Hian Kee

    2003-10-03

    Orthogonal array design (OAD) was applied for the first time to optimize microwave-assisted extraction (MAE) and supercritical fluid extraction (SFE) conditions for the analysis of four carbamates (propoxur, propham, methiocarb, chlorpropham) from soil. The theory and methodology of a new OA16 (4(4)) matrix derived from a OA16 (2(15)) matrix were developed during the MAE optimization. An analysis of variance technique was employed as the data analysis strategy in this study. Determinations of analytes were completed using high-performance liquid chromatography (HPLC) with UV detection. Four carbamates were successfully extracted from soil with recoveries ranging from 85 to 105% with good reproducibility (approximately 4.9% RSD) under the optimum MAE conditions: 30 ml methanol, 80 degrees C extraction temperature, and 6-min microwave heating. An OA8 (2(7)) matrix was employed for the SFE optimization. The average recoveries and RSD of the analytes from spiked soil by SFE were 92 and 5.5%, respectively except for propham (66.3+/-7.9%), under the following conditions: heating for 30 min at 60 degrees C under supercritical CO2 at 300 kg/cm2 modified with 10% (v/v) methanol. The composition of the supercritical fluid was demonstrated to be a crucial factor in the extraction. The addition of a small volume (10%) of methanol to CO2 greatly enhanced the recoveries of carbamates. A comparison of MAE with SFE was also conducted. The results indicated that >85% average recoveries were obtained by both optimized extraction techniques, and slightly higher recoveries of three carbamates (propoxur, propham and methiocarb) were achieved using MAE. SFE showed slightly higher recovery for chlorpropham (93 vs. 87% for MAE). The effects of time-aged soil on the extraction of analytes were examined and the results obtained by both methods were also compared.

  15. Distribution of six heavy metals in contaminated clay soils before and after extractive cleaning

    NARCIS (Netherlands)

    Tuin, B.J.W.; Tels, M.

    1990-01-01

    A sequential extraction procedure according to Tessier et al. is carried out to compare the distribution of six metals (Cd, Cr, Cu, Ni, Pb and Zn) in contaminated clay soils before and after extractive cleaning. Extraction of metals from the ‘soil fractions’ with 0.1 N HC1 or 0.1 M EDTA becomes more

  16. Simple practical approach for sample loading prior to DNA extraction using a silica monolith in a microfluidic device.

    Science.gov (United States)

    Shaw, Kirsty J; Joyce, Domino A; Docker, Peter T; Dyer, Charlotte E; Greenman, John; Greenway, Gillian M; Haswell, Stephen J

    2009-12-07

    A novel DNA loading methodology is presented for performing DNA extraction on a microfluidic system. DNA in a chaotropic salt solution was manually loaded onto a silica monolith orthogonal to the subsequent flow of wash and elution solutions. DNA was successfully extracted from buccal swabs using electro-osmotic pumping (EOP) coupled with in situ reagents contained within a 1.5% agarose gel matrix. The extracted DNA was of sufficient quantity and purity for polymerase chain reaction (PCR) amplification.

  17. Extraction of rare earth elements from a contaminated cropland soil using nitric acid, citric acid, and EDTA.

    Science.gov (United States)

    Tang, Hailong; Shuai, Weitao; Wang, Xiaojing; Liu, Yangsheng

    2017-08-01

    Rare earth elements (REEs) contamination to the surrounding soil has increased the concerns of health risk to the local residents. Soil washing was first attempted in our study to remediate REEs-contaminated cropland soil using nitric acid, citric acid, and ethylene diamine tetraacetic acid (EDTA) for soil decontamination and possible recovery of REEs. The extraction time, washing agent concentration, and pH value of the washing solution were optimized. The sequential extraction analysis proposed by Tessier was adopted to study the speciation changes of the REEs before and after soil washing. The extract containing citric acid was dried to obtain solid for the X-ray fluorescence (XRF) analysis. The results revealed that the optimal extraction time was 72 h, and the REEs extraction efficiency increased as the agent concentration increased from 0.01 to 0.1 mol/L. EDTA was efficient to extract REEs over a wide range of pH values, while citric acid was around pH 6.0. Under optimized conditions, the average extraction efficiencies of the major REEs in the contaminated soil were 70.96%, 64.38%, and 62.12% by EDTA, nitric acid, and citric acid, respectively. The sequential extraction analyses revealed that most soil-bounded REEs were mobilized or extracted except for those in the residual fraction. Under a comprehensive consideration of the extraction efficiency and the environmental impact, citric acid was recommended as the most suitable agent for extraction of the REEs from the contaminated cropland soils. The XRF analysis revealed that Mn, Al, Si, Pb, Fe, and REEs were the major elements in the extract indicating a possibile recovery of the REEs.

  18. Speciation of As(III), As(V), MMA and DMA in contaminated soil extracts by HPLC-ICP/MS

    Energy Technology Data Exchange (ETDEWEB)

    Bissen, M.; Frimmel, F.H. [Engler-Bunte-Institut, Univ. Karlsruhe (Germany)

    2000-05-01

    A method to separate and quantify two inorganic arsenic species As(III) and As(V) and two organic arsenic species, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), by HPLC-ICP/MS has been developed. The separation of arsenic species was achieved on the anionic exchange column IonPac {sup trademark} AS11 (Dionex) with NaOH as mobile phase. The technique was successfully applied to analyze extracts of two contaminated soils, sampled at a former tannery site (soil 1) and a former paint production site (soil 2). The soils were extracted at pH values similar to the natural environment. Extractions were performed at different pH values with 0.3 M ammonium oxalate (pH = 3), milli-Q water (pH = 5.8), 0.3 M sodium carbonate (pH = 8) and 0.3 M sodium bicarbonate (pH = 11). No organically bound arsenic was found in the extracts. As(V) was the major component. Only up to 0.04% of the total arsenic contained in soil 1 were mobilized. The highest amount of extracted arsenic was found at the highest pH. In the milli-Q water extract of soil 1 As(III) and As(V) were found. High amounts of As(V) were found in the extracts of soil 2. Up to 20% of the total arsenic bound to soil 2 constituents were released. The results show that the mobilization of arsenic depended on the pH value of the extraction solution and the kind of extracted soil. Dramatic consequences have to be expected for pH changes in the environment especially in cases where soils contain high amounts of mobile arsenic. (orig.)

  19. Determination of polyphenolic content, HPLC analyses and DNA cleavage activity of Malaysian Averrhoa carambola L. fruit extracts

    Directory of Open Access Journals (Sweden)

    Zakia Khanam

    2015-10-01

    Full Text Available In developing countries, the increasing gap between population growth and food supply has created renewed interest in finding reliable and cheap natural resources of nutraceutical value and health promoting properties. Therefore, the present study deals with the phytochemical analyses and DNA cleavage activity of Averrhoa carambola L. fruit (starfruit extracts. The phytochemical studies involve colour tests and quantification of phenolics and flavonoids of the prepared ethanolic and aqueous extracts. Identification of phenolic acids and flavonoids present in the extracts were conducted by high performance liquid chromatography (HPLC equipped with diode array detector (DAD. DNA cleavage activity of the extracts was evaluated through gel electrophoresis against plasmid Escherichia coli DNA at different concentrations (0.125–0.60 μg/μl. The results of the study exhibited that the starfruit is a rich source of polyphenols and all the extracts exhibited a dose dependent DNA cleavage activity, whereas ethanolic extract induced more cleavage as compared to the aqueous extract. In conclusion, the present study provides preliminary evidence with regard to nutraceutical value of the fruit. So, further extensive study is a prerequisite to exploit DNA cleaving properties of the fruit extracts for therapeutic application.

  20. Removal of petroleum-derived hydrocarbons from contaminated soils by solvent extraction

    International Nuclear Information System (INIS)

    Ladanowski, C.; Petti, L.

    1993-01-01

    Laboratory studies were conducted using hexane for the removal of light crude oil from contaminated sand, peat, and clay soils. The bench-scale process tested consists of three major steps: solvent washing, settling/decantation/filtration of extract, and solvent recycle. The results indicate that the use of solvent extraction for cleanup of oil-contaminated soils is an effective technology at the bench-scale level. Using a 1,000 g batch system, extremely high oil removal efficiencies were obtained from contaminated sand (up to 98.9%) and peat soil (up to 83.9%). The final oil contaminant concentration for sand varied between 0.06% and 0.39%, while that for peat soil varied between 1.52% and 5.21%. The guidelines for the decommissioning and cleanup of sites in Ontario for oil and grease (1 wt %) were met in all instances for the treated sand. Hexane recovery from diesel-contaminated sand and peat soil experiments was ca 81% and 67% respectively. 4 refs., 6 figs., 10 tabs

  1. Selenium speciation in phosphate mine soils and evaluation of a sequential extraction procedure using XAFS

    Energy Technology Data Exchange (ETDEWEB)

    Favorito, Jessica E.; Luxton, Todd P.; Eick, Matthew J.; Grossl, Paul R. (VP); (Utah SU); (EPA)

    2017-10-01

    Selenium is a trace element found in western US soils, where ingestion of Se-accumulating plants has resulted in livestock fatalities. Therefore, a reliable understanding of Se speciation and bioavailability is critical for effective mitigation. Sequential extraction procedures (SEP) are often employed to examine Se phases and speciation in contaminated soils but may be limited by experimental conditions. We examined the validity of a SEP using X-ray absorption spectroscopy (XAS) for both whole and a sequence of extracted soils. The sequence included removal of soluble, PO4-extractable, carbonate, amorphous Fe-oxide, crystalline Fe-oxide, organic, and residual Se forms. For whole soils, XANES analyses indicated Se(0) and Se(-II) predominated, with lower amounts of Se(IV) present, related to carbonates and Fe-oxides. Oxidized Se species were more elevated and residual/elemental Se was lower than previous SEP results from ICP-AES suggested. For soils from the SEP sequence, XANES results indicated only partial recovery of carbonate, Fe-oxide and organic Se. This suggests Se was incompletely removed during designated extractions, possibly due to lack of mineral solubilization or reagent specificity. Selenium fractions associated with Fe-oxides were reduced in amount or removed after using hydroxylamine HCl for most soils examined. XANES results indicate partial dissolution of solid-phases may occur during extraction processes. This study demonstrates why precautions should be taken to improve the validity of SEPs. Mineralogical and chemical characterizations should be completed prior to SEP implementation to identify extractable phases or mineral components that may influence extraction effectiveness. Sequential extraction procedures can be appropriately tailored for reliable quantification of speciation in contaminated soils.

  2. Two simple techniques for the safe Sarcoptes collection and individual mite DNA extraction.

    Science.gov (United States)

    Soglia, Dominga; Rambozzi, Luisa; Maione, Sandra; Spalenza, Veronica; Sartore, Stefano; Alasaad, Samer; Sacchi, Paola; Rossi, Luca

    2009-10-01

    Availability of mites is a recognized limiting factor of biological and genetic investigations of the genus Sarcoptes. Current methods of deoxyribonucleic acid (DNA) extraction from individual mites also need substantial improvement in efficiency and operator friendliness. We have first developed a technique for efficient and safe extraction of living mites from scabietic skin samples (crusts or deep skin scrapings). Its core device is a large plastic syringe connected with a 1.5-ml Eppendorf tube. The source material is introduced in the syringe and the device in a shoe box with the tip half of the tube emerging. Mites migrate towards a heat source during a minimum of 36 h. Then, the tube is detached and the mites utilized without risks for the operators. A second technique allows operator-friendly manipulation of individual mites for DNA extraction. Fixed mites are isolated by adhesion to a small strip of polyvinyl chloride (PVC) adhesive tape operated with tweezers. Then, mite and strip are plunged in the lyses buffer and the sample twice submitted to thermal shock for disruption of the chitinous exoskeleton. Data show that the tape does not interfere with successive DNA extraction with a commercial kit. The corresponding protocol, that we briefly name "PVC adhesive tape + thermal shock + kit DNA extraction," compares favorably with the available ones.

  3. Evaluation of five DNA extraction methods for purification of DNA from atherosclerotic tissue and estimation of prevalence of Chlamydia pneumoniae in tissue from a Danish population undergoing vascular repair

    Directory of Open Access Journals (Sweden)

    Lindholt Jes S

    2003-09-01

    Full Text Available Abstract Background To date PCR detection of Chlamydia pneumoniae DNA in atherosclerotic lesions from Danish patients has been unsuccessful. To establish whether non-detection was caused by a suboptimal DNA extraction method, we tested five different DNA extraction methods for purification of DNA from atherosclerotic tissue. Results The five different DNA extraction methods were tested on homogenate of atherosclerotic tissue spiked with C. pneumoniae DNA or EB, on pure C. pneumoniae DNA samples and on whole C. pneumoniae EB. Recovery of DNA was measured with a C. pneumoniae-specific quantitative real-time PCR. A DNA extraction method based on DNA-binding to spin columns with a silica-gel membrane (DNeasy Tissue kit showed the highest recovery rate for the tissue samples and pure DNA samples. However, an automated extraction method based on magnetic glass particles (MagNA Pure performed best on intact EB and atherosclerotic tissue spiked with EB. The DNeasy Tissue kit and MagNA Pure methods and the highly sensitive real-time PCR were subsequently used on 78 atherosclerotic tissue samples from Danish patients undergoing vascular repair. None of the samples were positive for C. pneumoniae DNA. The atherosclerotic samples were tested for inhibition by spiking with two different, known amounts of C. pneumoniae DNA and no samples showed inhibition. Conclusion As a highly sensitive PCR method and an optimised DNA extraction method were used, non-detection in atherosclerotic tissue from the Danish population was probably not caused by use of inappropriate methods. However, more samples may need to be analysed per patient to be completely certain on this. Possible methodological and epidemiological reasons for non-detection of C. pneumoniae DNA in atherosclerotic tissue from the Danish population are discussed. Further testing of DNA extraction methods is needed as this study has shown considerable intra- and inter-method variation in DNA recovery.

  4. Optimization of DNA extraction for RAPD and ISSR analysis of Arbutus unedo L. Leaves.

    Science.gov (United States)

    Sá, Olga; Pereira, José Alberto; Baptista, Paula

    2011-01-01

    Genetic analysis of plants relies on high yields of pure DNA. For the strawberry tree (Arbutus unedo) this represents a great challenge since leaves can accumulate large amounts of polysaccharides, polyphenols and secondary metabolites, which co-purify with DNA. For this specie, standard protocols do not produce efficient yields of high-quality amplifiable DNA. Here, we present for the first time an improved leaf-tissue protocol, based on the standard cetyl trimethyl ammonium bromide protocol, which yields large amounts of high-quality amplifiable DNA. Key steps in the optimized protocol are the addition of antioxidant compounds-namely polyvinyl pyrrolidone (PVP), 1,4-dithiothreitol (DTT) and 2-mercaptoethanol, in the extraction buffer; the increasing of CTAB (3%, w/v) and sodium chloride (2M) concentration; and an extraction with organic solvents (phenol and chloroform) with the incubation of samples on ice. Increasing the temperature for cell lyses to 70 °C also improved both DNA quality and yield. The yield of DNA extracted was 200.0 ± 78.0 μg/μL and the purity, evaluated by the ratio A(260)/A(280), was 1.80 ± 0.021, indicative of minimal levels of contaminating metabolites. The quality of the DNA isolated was confirmed by random amplification polymorphism DNA and by inter-simple sequence repeat amplification, proving that the DNA can be amplified via PCR.

  5. Optimization of DNA Extraction for RAPD and ISSR Analysis of Arbutus unedo L. Leaves

    Directory of Open Access Journals (Sweden)

    Paula Baptista

    2011-06-01

    Full Text Available Genetic analysis of plants relies on high yields of pure DNA. For the strawberry tree (Arbutus unedo this represents a great challenge since leaves can accumulate large amounts of polysaccharides, polyphenols and secondary metabolites, which co-purify with DNA. For this specie, standard protocols do not produce efficient yields of high-quality amplifiable DNA. Here, we present for the first time an improved leaf-tissue protocol, based on the standard cetyl trimethyl ammonium bromide protocol, which yields large amounts of high-quality amplifiable DNA. Key steps in the optimized protocol are the addition of antioxidant compounds—namely polyvinyl pyrrolidone (PVP, 1,4-dithiothreitol (DTT and 2-mercaptoethanol, in the extraction buffer; the increasing of CTAB (3%, w/v and sodium chloride (2M concentration; and an extraction with organic solvents (phenol and chloroform with the incubation of samples on ice. Increasing the temperature for cell lyses to 70 °C also improved both DNA quality and yield. The yield of DNA extracted was 200.0 ± 78.0 µg/µL and the purity, evaluated by the ratio A260/A280, was 1.80 ± 0.021, indicative of minimal levels of contaminating metabolites. The quality of the DNA isolated was confirmed by random amplification polymorphism DNA and by inter-simple sequence repeat amplification, proving that the DNA can be amplified via PCR.

  6. Detailed screening of the soil faunal diversity using a tiered DNA metabarcoding approach

    DEFF Research Database (Denmark)

    Groot, G.A. de; Geisen, S.; Costa, D.

    is not a realistic proposition. DNA-based approaches, especially high-throughput DNA metabarcoding assays, potentially solve this issue, but the development of such methods targeting soil fauna lags far behind that of soil microbes. Within the EU FP7-project EcoFINDERS, we developed and tested a framework...... analyzed to obtain high resolution data for six different groups: mites, collembola, enchytraeids, nematodes, earthworms and protists. New primer sets, as well as reference barcode datasets were established for several of them. Here, we show the results of two test runs based on 454 pyrosequencing....... In the first run, artificially created DNA pools of known composition were analysed to test to which extent the taxonomic composition could successfully be retrieved. Preliminary results show that for all groups the majority of species in the DNA pool were recovered by the metabarcoding approach. By comparing...

  7. A high throughput DNA extraction method with high yield and quality

    Directory of Open Access Journals (Sweden)

    Xin Zhanguo

    2012-07-01

    Full Text Available Abstract Background Preparation of large quantity and high quality genomic DNA from a large number of plant samples is a major bottleneck for most genetic and genomic analyses, such as, genetic mapping, TILLING (Targeting Induced Local Lesion IN Genome, and next-generation sequencing directly from sheared genomic DNA. A variety of DNA preparation methods and commercial kits are available. However, they are either low throughput, low yield, or costly. Here, we describe a method for high throughput genomic DNA isolation from sorghum [Sorghum bicolor (L. Moench] leaves and dry seeds with high yield, high quality, and affordable cost. Results We developed a high throughput DNA isolation method by combining a high yield CTAB extraction method with an improved cleanup procedure based on MagAttract kit. The method yielded large quantity and high quality DNA from both lyophilized sorghum leaves and dry seeds. The DNA yield was improved by nearly 30 fold with 4 times less consumption of MagAttract beads. The method can also be used in other plant species, including cotton leaves and pine needles. Conclusion A high throughput system for DNA extraction from sorghum leaves and seeds was developed and validated. The main advantages of the method are low cost, high yield, high quality, and high throughput. One person can process two 96-well plates in a working day at a cost of $0.10 per sample of magnetic beads plus other consumables that other methods will also need.

  8. Comparative effects of several cyclodextrins on the extraction of PAHs from an aged contaminated soil

    International Nuclear Information System (INIS)

    Sánchez-Trujillo, M.A.; Morillo, E.; Villaverde, J.; Lacorte, S.

    2013-01-01

    The objective of the present study was to characterise the polycyclic aromatic hydrocarbons (PAHs) content of an aged contaminated soil and to propose remediation techniques using cyclodextrins (CDs). Four CDs solutions were tested as soil decontamination tool and proved more efficient in extracting PAHs than when an aqueous solution was used; especially two chemically modified CDs resulted in higher extraction percentages than natural β-CD. The highest extraction percentages were obtained for 3-ring PAHs, because of the appropriate size and shape of these compounds relative to those of the hydrophobic cavities of the CDs studied. A detailed mechanistic interpretation of the chemical modification of CDs on the extraction of the different PAHs has been performed, and connected with the role that the different hydrophobicities of the PAHs play in the extraction behaviour observed for the 16 PAHs, limiting their accessibility and the remaining risk of those PAHs not extractable by CDs. -- Highlights: ► Four cyclodextrins (CDs) solutions were tested as soil decontamination tool for PAHs. ► Extractions with CDs were higher than with electrolyte, especially with synthetic CDs. ► Extraction capacity depends on the adequate size of PAHs and CDs hydrophobic cavity. ► 2–3 ring PAHs, the more abundant in the soil, were extracted in higher percentages. ► CDs extract preferably the less hydrophobic and more potentially toxic PAHs. -- Cyclodextrin solutions are useful and interesting tools for the decontamination of soils polluted by PAHs

  9. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types

    DEFF Research Database (Denmark)

    Lever, Mark; Torti, Andrea; Eickenbusch, Philip

    2015-01-01

    tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world...... DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample...

  10. Remediation of sandy soils contaminated with hydrocarbons and halogenated hydrocarbons by soil vapour extraction.

    Science.gov (United States)

    Albergaria, José Tomás; Alvim-Ferraz, Maria da Conceição M; Delerue-Matos, Cristina

    2012-08-15

    This paper presents the study of the remediation of sandy soils containing six of the most common contaminants (benzene, toluene, ethylbenzene, xylene, trichloroethylene and perchloroethylene) using soil vapour extraction (SVE). The influence of soil water content on the process efficiency was evaluated considering the soil type and the contaminant. For artificially contaminated soils with negligible clay contents and natural organic matter it was concluded that: (i) all the remediation processes presented efficiencies above 92%; (ii) an increase of the soil water content led to a more time-consuming remediation; (iii) longer remediation periods were observed for contaminants with lower vapour pressures and lower water solubilities due to mass transfer limitations. Based on these results an easy and relatively fast procedure was developed for the prediction of the remediation times of real soils; 83% of the remediation times were predicted with relative deviations below 14%. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Evaluation of a manual DNA extraction protocol and an isothermal amplification assay for detecting HIV-1 DNA from dried blood spots for use in resource-limited settings.

    Science.gov (United States)

    Jordan, Jeanne A; Ibe, Christine O; Moore, Miranda S; Host, Christel; Simon, Gary L

    2012-05-01

    In resource-limited settings (RLS) dried blood spots (DBS) are collected on infants and transported through provincial laboratories to a central facility where HIV-1 DNA PCR testing is performed using specialized equipment. Implementing a simpler approach not requiring such equipment or skilled personnel could allow the more numerous provincial laboratories to offer testing, improving turn-around-time to identify and treat infected infants sooner. Assess performances of a manual DNA extraction method and helicase-dependent amplification (HDA) assay for detecting HIV-1 DNA from DBS. 60 HIV-1 infected adults were enrolled, blood samples taken and DBS made. DBS extracts were assessed for DNA concentration and beta globin amplification using PCR and melt-curve analysis. These same extracts were then tested for HIV-1 DNA using HDA and compared to results generated by PCR and pyrosequencing. Finally, HDA limit of detection (LOD) studies were performed using DBS extracts prepared with known numbers of 8E5 cells. The manual extraction protocol consistently yielded high concentrations of amplifiable DNA from DBS. LOD assessment demonstrated HDA detected ∼470 copies/ml of HIV-1 DNA extracts in 4/4 replicates. No statistical difference was found using the McNemar's test when comparing HDA to PCR for detecting HIV-1 DNA from DBS. Using just a magnet, heat block and pipettes, the manual extraction protocol and HDA assay detected HIV-1 DNA from DBS at levels that would be useful for early infant diagnosis. Next steps will include assessing HDA for non-B HIV-1 subtypes recognition and comparison to Roche HIV-1 DNA v1.5 PCR assay. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae).

    Science.gov (United States)

    Zhao, Ya-E; Xu, Ji-Ru; Hu, Li; Wu, Li-Ping; Wang, Zheng-Hang

    2012-05-01

    The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900 bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples ( ≥ 1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. {sup 32}P-postlabeling determination of DNA adducts in the earthworm Lumbricus terrestris exposed to PAH-contaminated soils

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, P. [Laval Univ. Research Center, Quebec (Canada)]|[Ministere de l`Environnement et de la Faune du Quebec (Canada); El Adlouni, C.; Mukhopadhyay, M.J.; Nadeau, D.; Poirier, G.G. [Laval Univ. Research Center, Quebec (Canada); Viel, G. [CreaLab., Quebec (Canada)

    1995-05-01

    The importance of the search for reliable biomarkers of DNA damage in environmental health assessment is well recognized by the scientific community and regulatory agencies. Among the major biomarkers of DNA damage is the measurement of DNA adducts in target cells or tissues. Up to now, DNA adduct determinations have been directed mostly toward human exposure to toxic substances from the workplace and environment. Moreover, techniques for measuring DNA adducts, and in particular the {sup 32}P-postlabelling technique, presented also the possibility of determining DNA adduct levels in endogenous animal populations exposed to polluted environments as early warning monitors of ecotoxicity. Soil contamination is becoming a major environmental issue. Therefore, numerous contaminated sites must now be remediated to protect human health and to permit new uses of these sites as agricultural, residential, or industrial areas. Fulfillment of this task requires standardized and sensitive bioassays to carry out site evaluations and to establish scientifically defensible soil quality criteria. To that effect, the earthworm appears to be one of the best organisms for use in soil toxicity evaluation. Earthworms are probably the most relevant soil species, representing 60 to 80% of the total animal biomass in soil. Present soil bioassays focus mostly on plant species with end points like seed germination, root elongation, seedling growth and seedling emergence, and on acute toxicity evaluation (re: LC 50) on the earthworm Eisenia fetida. As yet, a standardized soil invertebrate test for teratogenic or mutagenic end points has not been developed. In this paper, we report the feasibility of DNA adduct determination by {sup 32}P-postlabelling in the earthworm Lumbricus terrestris as a way to detect the presence of genotoxic substances in soils. 20 refs., 1 fig., 1 tab.

  14. Establishing a novel automated magnetic bead-based method for the extraction of DNA from a variety of forensic samples.

    Science.gov (United States)

    Witt, Sebastian; Neumann, Jan; Zierdt, Holger; Gébel, Gabriella; Röscheisen, Christiane

    2012-09-01

    Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO(®)150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch(®)Forensic DNA Purification Kit (Invitrogen), the PrepFiler™Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  15. The status of phosphorus in Thai soils and P evaluation using EDTA-NaF extraction method

    Directory of Open Access Journals (Sweden)

    Toru Matoh

    2003-07-01

    Full Text Available Although the available P extracted by Bray II method in tropical soil is low, most of tropical plants can grow well. The objective of this study was to study P status and to evaluate the available P extracted by EDTA-NaF method. Top soil and sub soil of 10 dominant soil series in Thailand were analyzed for some chemical properties and characterization of the forms of phosphorus using EDTA-NaF extraction and successive phosphorus extraction by the modified Sekiya method. The soil total P concentration was 38-1137 mg P2O5 kg-1. The available Bray II-P was very low to high (1-76 mg P2O5 kg-1, and it approximated 0.17-12% of the total P. Iron and aluminum phosphates were the main fraction of inorganic P in acid soil, whereas Ca phosphates were in calcareous soils. Organic P content accounted for 33-67% and most of them were bound with Fe and Al in acid soils and Ca in calcareous soils. P extracted by EDTA-NaF reagent was obviously larger than that of Bray II reagent. The EDTA-NaF extracted P [high molecular weight organic P (HMWP+ inorganic P (EDTA ext Pi] was 7-46% and 1-6% of total P in acid soils and calcareous soils respectively. The EDTA ext Pi tended to be larger than HMWP except in Tk soil. The total amount of extracted P correlated well with Al-Pi and Fe-Pi which were the main fraction of inorganic P. It also correlated with HMWP, but HMWP did not correlate with organic P determine by ignition method and Ca-Po, Fe-Po and Al-Po. The EDTA-NaF method may be suitable for P evaluation in the soils which have high amounts of Fe-Pi, Al -Pi and organic P widely distributed in Thailand.

  16. Hot water extraction with in situ wet oxidation: Kinetics of PAHs removal from soil

    International Nuclear Information System (INIS)

    Dadkhah, Ali A.; Akgerman, Aydin

    2006-01-01

    Finding environmentally friendly and cost-effective methods to remediate soils contaminated with polycyclic aromatic hydrocarbons (PAHs) is currently a major concern of researchers. In this study, a series of small-scale semi-continuous extractions - with and without in situ wet oxidation - were performed on soils polluted with PAHs, using subcritical water (i.e. liquid water at high temperatures and pressures, but below the critical point) as the removal agent. Experiments were performed in a 300 mL reactor using an aged soil sample. To find the desorption isotherms and oxidation reaction rates, semi-continuous experiments with residence times of 1 and 2 h were performed using aged soil at 250 deg. C and hydrogen peroxide as oxidizing agent. In all combined extraction and oxidation flow experiments, PAHs in the remaining soil after the experiments were almost undetectable. In combined extraction and oxidation no PAHs could be detected in the liquid phase after the first 30 min of the experiments. Based on these results, extraction with hot water, if combined with oxidation, should reduce the cost of remediation and can be used as a feasible alternative technique for remediating contaminated soils and sediments

  17. Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.

    Directory of Open Access Journals (Sweden)

    Gemma Henderson

    Full Text Available Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However

  18. Dichlorvos exposure impedes extraction and amplification of DNA from insects in museum collections

    Directory of Open Access Journals (Sweden)

    Åkerlund Monika

    2010-01-01

    Full Text Available Abstract Background The insecticides dichlorvos, paradichlorobenzene and naphthalene have been commonly used to eradicate pest insects from natural history collections. However, it is not known how these chemicals affect the DNA of the specimens in the collections. We thus tested the effect of dichlorvos, paradichlorobenzene and naphthalene on DNA of insects (Musca domestica by extracting and amplifying DNA from specimens exposed to insecticides in two different concentrations over increasing time intervals. Results The results clearly show that dichlorvos impedes both extraction and amplification of mitochondrial and nuclear DNA after relatively short time, whereas paradichlorobenzene and naphthalene do not. Conclusion Collections treated with paradichlorobenzene and naphthalene, are better preserved concerning DNA, than those treated with dichlorvos. Non toxic pest control methods should, however, be preferred due to physical damage of specimens and putative health risks by chemicals.

  19. Organic acid enhanced electrodialytic extraction of lead from contaminated soil fines in suspension

    DEFF Research Database (Denmark)

    Jensen, Pernille Erland; Ahring, Birgitte Kiær; Ottosen, Lisbeth M.

    2007-01-01

    for decontamination of the sludge was investigated. The ability of 11 organic acids to extract Pb from the fine fraction of contaminated soil (grains soil fines in suspension......The implementation of soil washing technology for the treatment of heavy metal contaminated soils is limited by the toxicity and unwieldiness of the remaining heavy metal contaminated sludge. In this work, the feasibility of combining electrodialytic remediation with heterotrophic leaching...... was tested. Five of the acids showed the ability to extract Ph from the soil fines in excess of the effect caused solely by pH changes. Addition of the acids, however, severely impeded EDR, hence promotion of EDR by combination with heterotrophic leaching was rejected. In contrast, enhancement of EDR...

  20. Benzo(a)pyrene accumulation in soils of technogenic emission zone by subcritical water extraction method

    Science.gov (United States)

    Sushkova, Svetlana; Minkina, Tatiana; Kizilkaya, Ridvan; Mandzhieva, Saglara; Batukaev, Abdulmalik; Bauer, Tatiana; Gulser, Coskun

    2016-04-01

    The purpose of research is the assessment of main marker of polycyclic aromatic hydrocarbons contamination, benzo[a]pyrene (BaP) content in soils of emission zone of the power complex plant in soils with use of ecologically clean and effective subcritical water extraction method. Studies were conducted on the soils of monitoring plots subjected to Novocherkassk Power Plant emissions from burning coal. In 2000, monitoring plots were established at different distances from the NPS (1.0-20.0 km). Soil samples for the determination of soil properties and the contents of BaP were taken from a depth of 0-20 cm. The soil cover in the region under study consisted of ordinary chernozems, meadow-chernozemic soils, and alluvial meadow soils. This soil revealed the following physical and chemical properties: Corg-3.1-5.0%, pH-7.3-7.6, ECE-31.2-47.6 mmol(+)/100g; CaCO3-0.2-1.0%, the content of physical clay - 51-67% and clay - 3-37%. BaP extraction from soils was carried out by a subcritical water extraction method. Subcritical water extraction of BaP from soil samples was conducted in a specially developed extraction cartridge made of stainless steel and equipped with screw-on caps at both ends. It was also equipped with a manometer that included a valve for pressure release to maintain an internal pressure of 100 atm. The extraction cartridge containing a sample and water was placed into an oven connected to a temperature regulator under temperature 250oC and pressure 60 atm. The BaP concentration in the acetonitrile extract was determined by HPLC. The efficiency of BaP extraction from soil was determined using a matrix spike. The main accumulation of pollutant in 20 cm layer of soils is noted directly in affected zone on the plots situated at 1.2, 1.6, 5.0, 8.0 km from emission source in the direction of prevailing winds. The maximum quantity of a pollutant was founded in the soil of the plot located mostly close to a source of pollution in the direction of prevailing winds

  1. Polycyclic aromatic hydrocarbons bioavailability in industrial and agricultural soils: Linking SPME and Tenax extraction with bioassays.

    Science.gov (United States)

    Guo, Meixia; Gong, Zongqiang; Li, Xiaojun; Allinson, Graeme; Rookes, James; Cahill, David

    2017-06-01

    The aims of this study were to evaluate the bioavailability of polycyclic aromatic hydrocarbons (PAHs) in industrial and agricultural soils using chemical methods and a bioassay, and to study the relationships between the methods. This was conducted by comparing the quantities of PAHs extracted from two manufactured gas plant (MGP) soils and an agricultural soil with low level contamination by solid-phase micro-extraction (SPME) and Tenax-TA extraction with the quantities taken up by the earthworm (Eisenia fetida). In addition, a biodegradation experiment was conducted on one MGP soil (MGP-A) to clarify the relationship between PAH removal by biodegradation and the variation in PAH concentrations in soil pore water. Results demonstrated that the earthworm bioassay could not be used to examine PAH bioavailability in the tested MGP soils; which was the case even in the diluted MGP-A soils after biodegradation. However, the bioassay was successfully applied to the agricultural soil. These results suggest that earthworms can only be used for bioassays in soils with low toxicity. In general, rapidly desorbing concentrations extracted by Tenax-TA could predict PAH concentrations accumulated in earthworms (R 2 =0.66), while SPME underestimated earthworm concentrations by a factor of 2.5. Both SPME and Tenax extraction can provide a useful tool to predict PAH bioavailability for earthworms, but Tenax-TA extraction was proven to be a more sensitive and precise method than SPME for the prediction of earthworm exposure in the agricultural soil. Copyright © 2017. Published by Elsevier Inc.

  2. Optimization of HPV DNA detection in urine by improving collection, storage, and extraction.

    Science.gov (United States)

    Vorsters, A; Van den Bergh, J; Micalessi, I; Biesmans, S; Bogers, J; Hens, A; De Coster, I; Ieven, M; Van Damme, P

    2014-11-01

    The benefits of using urine for the detection of human papillomavirus (HPV) DNA have been evaluated in disease surveillance, epidemiological studies, and screening for cervical cancers in specific subgroups. HPV DNA testing in urine is being considered for important purposes, notably the monitoring of HPV vaccination in adolescent girls and young women who do not wish to have a vaginal examination. The need to optimize and standardize sampling, storage, and processing has been reported.In this paper, we examined the impact of a DNA-conservation buffer, the extraction method, and urine sampling on the detection of HPV DNA and human DNA in urine provided by 44 women with a cytologically normal but HPV DNA-positive cervical sample. Ten women provided first-void and midstream urine samples. DNA analysis was performed using real-time PCR to allow quantification of HPV and human DNA.The results showed that an optimized method for HPV DNA detection in urine should (a) prevent DNA degradation during extraction and storage, (b) recover cell-free HPV DNA in addition to cell-associated DNA, (c) process a sufficient volume of urine, and (d) use a first-void sample.In addition, we found that detectable human DNA in urine may not be a good internal control for sample validity. HPV prevalence data that are based on urine samples collected, stored, and/or processed under suboptimal conditions may underestimate infection rates.

  3. Evaluation and optimization of nucleic acid extraction methods for the molecular analysis of bacterial communities associated with corrored steel

    NARCIS (Netherlands)

    Marty, F.; Ghiglione, J.-F.; Païsse, S.; Gueuné, H.; Quillet, L.; van Loosdrecht, M.C.M.; Muyzer, G.

    2012-01-01

    Different DNA and RNA extraction approaches were evaluated and protocols optimized on in situ corrosion products from carbon steel in marine environments. Protocols adapted from the PowerSoil DNA/RNA Isolation methods resulted in the best nucleic acid (NA) extraction performances (ie combining high

  4. The effects of exotic and native poplars on rhizosphere soil microbe ...

    African Journals Online (AJOL)

    user

    2012-01-19

    Jan 19, 2012 ... ecology. However, how different poplars species affect soil microbial community and soil .... ml of molasses-urea block (MUB) (pH 6.0) and 0.5 ml of 25 mM p- ... Microbial community DNA extraction and polymerase chain.

  5. Soil sampling and extraction methods with possible application to pear thrips (Thysanoptera: Thripidae)

    Science.gov (United States)

    John E. Bater

    1991-01-01

    Techniques are described for the sampling and extraction of microarthropods from soil and the potential of these methods to extract the larval stages of the pear thrips, Taeniothrips inconsequens (Uzel), from soil cores taken in sugar maple stands. Also described is a design for an emergence trap that could be used to estimate adult thrips...

  6. Comparison of Soxhlet and Shake Extraction of Polycyclic Aromatic Hydrocarbons from Coal Tar Polluted Soils Sampled in the Field

    DEFF Research Database (Denmark)

    Lindhardt, Bo; Holst, Helle; Christensen, Thomas Højlund

    1994-01-01

    This study compares three extraction methods for PAHs in coal tar polluted soil: 3-times repeated shaking of the soil with dichloromethane-methanol (1:1), Soxhlet extraction with dichloromethane, and Soxhlet extraction with dichloromethane followed by Soxhlet extraction with methanol....... The extraction efficiencies were determined for ten selected PAHs in triplicate samples of six soils sampled at former gasworks sites. The samples covered a wide range of PAH concentrations, from 0.6 to 397 mg/kg soil. Soxhlet extraction with dichloromethane followed by Soxhlet extraction with methanol...

  7. Comparison of nine DNA extraction methods for the diagnosis of bovine tuberculosis by real time PCR

    Directory of Open Access Journals (Sweden)

    André Moura

    2016-07-01

    Full Text Available ABSTRACT: Bovine tuberculosis is an infectious disease with a high impact on the cattle industry, particularly in developing countries. PCR is a very sensitive method for detection of infectious agents, but the sensitivity of molecular diagnosis is largely dependent on the efficiency of the DNA extraction methods. The objective of this study was to evaluate DNA extraction methods for direct detection of Mycobacterium bovis in bovine tissue. Nine commercial kits for DNA extraction were evaluated when combined with two real time PCRs. The DNeasy Blood & Tissue Kit from QIAGEN showed better performance and sensitivity followed by the DNA Mini Kit RBC and FTA Elute Micro Card. Results suggested that, even when the analytical sensitivity of the qPCR is very high, the extraction method can influence the diagnostic sensitivity.

  8. DNA-methylation changes induced by salt stress in wheat Triticum ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-11-16

    Nov 16, 2009 ... soil roots are the primary point of contact with ionic toxicity and osmotic stress. One of .... liquid nitrogen with mortar and pestle. Then 10 ml of ... room temperature and the crude DNA extract was mixed with 10 ml of chloroform: ..... induces DNA hypomethylation, as reviewed by Cerda and. Weitzman (1997).

  9. Grape (Vitis vinifera) extracts protect against radiation-induced oxidative stress and DNA damage

    International Nuclear Information System (INIS)

    Singha, Indrani; Das, Subir Kumar; Saxena, S.; Gautam, S.

    2016-01-01

    Ionizing radiation (IR) causes oxidative stress through the overwhelming generation of reactive oxygen species (ROS) in the living cells leading further to the oxidative damage to biomolecules. Grapes (Vitis vinifera) contain several bioactive phytochemicals and are the richest source of antioxidant. In this study, we investigated and compared in vitro antioxidant activity and DNA damage protective property of the grape extracts of four different cultivars, including the Thompson seedless, Flame seedless, Kishmish chorni and Red globe. The activities of ascorbic acid oxidase and catalase significantly (p<0.01) differed among extracts within the same cultivar, while that of peroxidase and polyphenol oxidase did not differ significantly among extracts of any cultivar. In vitro antioxidant activities were assessed by ferric-reducing antioxidant power (FRAP) assay and ABTS. The superoxide radical-scavenging activity was higher in the seed as compared to the skin or pulp of the same cultivar. DNA damage was evaluated in acellular system using pBR322 plasmid relaxation. Grape extract was able to effectively scavenge free radicals in vitro. It could significantly prevent radiation-induced DNA damage. Furthermore, the protective action of grape depends on the source of extract and type of the cultivars. (author)

  10. Extraction and analysis of 14C-carbofuran radioactivity in soil sample

    International Nuclear Information System (INIS)

    Maizatul Akmam Mhd Nasir; Nashriyah Mat

    2005-01-01

    Carbofuran insecticide or nematicide sprayed onto soil in the agroecosystem will be taken up by plant. Carbofuran residue will pollute the environment and organisms in the food chain. Extraction and analysis of 14 C-carbofuran in soil from lysimeter were carried out. The Liquid Scintillation Counter (LSC) was used to measure radioactivity of 14 C-carbofuran in soil sample. (Author)

  11. Formation and release of non-extractable 14C-Dicamba residues in soil under sterile and non-sterile regimes

    International Nuclear Information System (INIS)

    Gevao, Bondi; Jones, Kevin C.; Semple, Kirk T.

    2005-01-01

    The role of native soil microorganisms in the formation and release of non-extractable 14 C-residues, previously treated with 14 C-Dicamba, was investigated to examine their significance to the longer-term environmental effects on non-extractable pesticide residues. A 90 d study compared the fate of Dicamba under sterile and non-sterile regimes. In addition, soils were aged for 30 d and repeatedly extracted with a 0.01 M CaCl 2 solution, to an extraction end point, to produce non-extractable residues. The extracted soil containing non-extractable residues was mixed with clean soil that had been freshly spiked with non-labeled Dicamba at 0.2 mg kg -1 to increase the bulk volume of the soil and stimulate microbial activity. Sub-samples were then introduced into microcosms to compare the extent of microbially facilitated release and mineralisation with release rates in sterile microcosms. The results show that microorganisms play a significant role in the formation and release of non-extractable Dicamba residues. The release of 14 C-activity in sterile microcosms was linked to physical mixing of the extracted soil with field soil prior to the beginning of the incubations. The released 14 C-activity may be further mineralized, reincorporated into humus, or taken up by plants or other soil inhabiting biota

  12. Model analysis of mechanisms controlling pneumatic soil vapor extraction

    DEFF Research Database (Denmark)

    Høier, Camilla Kruse; Sonnenborg, Torben Obel; Jensen, Karsten Høgh

    2009-01-01

    of heterogeneous soils by enforcing large fluctuating pressure fronts through the contaminated area. Laboratory experiments have suggested that pneumatic SVE considerably improves the recovery rate from low-permeable units. We have analyzed the experimental results using a numerical code and quantified......The efficiency of traditional soil venting or soil vapor extraction (SVE) highly depends on the architecture of the subsurface because imposed advective air flow tends to bypass low-permeable contaminated areas. Pneumatic SVE is a technique developed to enhance remediation efficiency...... level the pneumatic venting technology is superior to the traditional technique, and that the method is particularly efficient in cases where large permeability contrasts exist between soil units in the subsurface....

  13. Comparison of eight methods for the extraction of Bacillus atrophaeus spore DNA from eleven common interferents and a common swab.

    Directory of Open Access Journals (Sweden)

    Helen L Rose

    Full Text Available Eight DNA extraction products or methods (Applied Biosystems PrepFiler Forensic DNA Extraction Kit; Bio-Rad Instagene Only, Bio-Rad Instagene & Spin Column Purification; EpiCentre MasterPure DNA & RNA Kit; FujiFilm QuickGene Mini80; Idaho Technologies 1-2-3 Q-Flow Kit; MoBio UltraClean Microbial DNA Isolation Kit; Sigma Extract-N-Amp Plant and Seed Kit were adapted to facilitate extraction of DNA under BSL3 containment conditions. DNA was extracted from 12 common interferents or sample types, spiked with spores of Bacillus atropheaus. Resulting extracts were tested by real-time PCR. No one method was the best, in terms of DNA extraction, across all sample types. Statistical analysis indicated that the PrepFiler method was the best method from six dry powders (baking, biological washing, milk, plain flour, filler and talcum and one solid (Underarm deodorant, the UltraClean method was the best from four liquids (aftershave, cola, nutrient broth, vinegar, and the MasterPure method was the best from the swab sample type. The best overall method, in terms of DNA extraction, across all sample types evaluated was the UltraClean method.

  14. Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding, genotyping, and disease diagnostics from fungal and oomycete samples.

    Science.gov (United States)

    Osmundson, Todd W; Eyre, Catherine A; Hayden, Katherine M; Dhillon, Jaskirn; Garbelotto, Matteo M

    2013-01-01

    The ubiquity, high diversity and often-cryptic manifestations of fungi and oomycetes frequently necessitate molecular tools for detecting and identifying them in the environment. In applications including DNA barcoding, pathogen detection from plant samples, and genotyping for population genetics and epidemiology, rapid and dependable DNA extraction methods scalable from one to hundreds of samples are desirable. We evaluated several rapid extraction methods (NaOH, Rapid one-step extraction (ROSE), Chelex 100, proteinase K) for their ability to obtain DNA of quantity and quality suitable for the following applications: PCR amplification of the multicopy barcoding locus ITS1/5.8S/ITS2 from various fungal cultures and sporocarps; single-copy microsatellite amplification from cultures of the phytopathogenic oomycete Phytophthora ramorum; probe-based P. ramorum detection from leaves. Several methods were effective for most of the applications, with NaOH extraction favored in terms of success rate, cost, speed and simplicity. Frozen dilutions of ROSE and NaOH extracts maintained PCR viability for over 32 months. DNA from rapid extractions performed poorly compared to CTAB/phenol-chloroform extracts for TaqMan diagnostics from tanoak leaves, suggesting that incomplete removal of PCR inhibitors is an issue for sensitive diagnostic procedures, especially from plants with recalcitrant leaf chemistry. NaOH extracts exhibited lower yield and size than CTAB/phenol-chloroform extracts; however, NaOH extraction facilitated obtaining clean sequence data from sporocarps contaminated by other fungi, perhaps due to dilution resulting from low DNA yield. We conclude that conventional extractions are often unnecessary for routine DNA sequencing or genotyping of fungi and oomycetes, and recommend simpler strategies where source materials and intended applications warrant such use. © 2012 Blackwell Publishing Ltd.

  15. High-Quality and -Quantity DNA Extraction from Frozen Archival Blood Clots for Genotyping of Single-Nucleotide Polymorphisms

    DEFF Research Database (Denmark)

    Bank, Steffen; Nexø, Bjørn Andersen; Andersen, Vibeke

    2013-01-01

    the efficiency of commercial purification kits for extracting DNA from long-term frozen clotted blood. Methods: Serum tubes with clotted blood were stored at −20°C for 1 to 2.5 years before DNA extraction. DNA was extracted from 10 blood clot samples using PureGene (Qiagen) with and without glycogen, the QIAamp...... with a median of 0.65 μg (range 0.5–2.6 μg) pr 300 μL total blood. Conclusion: The yield obtained by the different commercial kits varied considerably. Our work demonstrates that high-quality and -quantity DNA can be extracted with the Maxwell 16 Blood purification kit (Promega) from cryopreserved blood clots...

  16. Evaluation and comparison of FTA card and CTAB DNA extraction methods for non-agricultural taxa1

    Science.gov (United States)

    Siegel, Chloe S.; Stevenson, Florence O.; Zimmer, Elizabeth A.

    2017-01-01

    Premise of the study: An efficient, effective DNA extraction method is necessary for comprehensive analysis of plant genomes. This study analyzed the quality of DNA obtained using paper FTA cards prepared directly in the field when compared to the more traditional cetyltrimethylammonium bromide (CTAB)–based extraction methods from silica-dried samples. Methods: DNA was extracted using FTA cards according to the manufacturer’s protocol. In parallel, CTAB-based extractions were done using the automated AutoGen DNA isolation system. DNA quality for both methods was determined for 15 non-agricultural species collected in situ, by gel separation, spectrophotometry, fluorometry, and successful amplification and sequencing of nuclear and chloroplast gene markers. Results: The FTA card extraction method yielded less concentrated, but also less fragmented samples than the CTAB-based technique. The card-extracted samples provided DNA that could be successfully amplified and sequenced. The FTA cards are also useful because the collected samples do not require refrigeration, extensive laboratory expertise, or as many hazardous chemicals as extractions using the CTAB-based technique. Discussion: The relative success of the FTA card method in our study suggested that this method could be a valuable tool for studies in plant population genetics and conservation biology that may involve screening of hundreds of individual plants. The FTA cards, like the silica gel samples, do not contain plant material capable of propagation, and therefore do not require permits from the U.S. Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) for transportation. PMID:28224056

  17. Technical reproducibility of single-nucleotide and size-based DNA biomarker assessment using DNA extracted from formalin-fixed, paraffin-embedded tissues.

    Science.gov (United States)

    Zhang, Shenli; Tan, Iain B; Sapari, Nur S; Grabsch, Heike I; Okines, Alicia; Smyth, Elizabeth C; Aoyama, Toru; Hewitt, Lindsay C; Inam, Imran; Bottomley, Dan; Nankivell, Matthew; Stenning, Sally P; Cunningham, David; Wotherspoon, Andrew; Tsuburaya, Akira; Yoshikawa, Takaki; Soong, Richie; Tan, Patrick

    2015-05-01

    DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5' untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples and 34 DNAs extracted from fresh-frozen tissues and cell lines. Each polymorphism was analyzed by two independent runs. We found that iPLEX biomarker assays measuring single-nucleotide polymorphisms provided consistent concordant results. However, by using FFPE DNA, size-based PCR biomarkers (GSTT1 and TYMS 5' untranslated region) were discrepant in 32.7% (16/49, with exact 95% CI, 19.9%-47.5%; exact binomial confidence limit test) and 4.2% (2/48, with exact 95% CI, 0.5%-14.3%) of cases, respectively, whereas no discrepancies were observed using intact genomic DNA. Our findings suggest that DNA from FFPE material can be used to reliably test single-nucleotide polymorphisms. However, results based on size-based PCR biomarkers, and particularly GSTT1 deletions, using FFPE DNA need to be interpreted with caution. Independent repeated assays should be performed on all cases to assess potential discrepancies. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  18. Phyto extraction Of Cadmium And Zinc From Contaminated Soils

    International Nuclear Information System (INIS)

    Lotfy, S.M.; Mostafa, A.Z.; Abdel Sabour, M. F.

    2012-01-01

    A trial was made to study the use of different plant species to extract heavy metals out of contaminated soils. Four Kg of each air-dried surface soil sample (0-20 cm) were packed in plastic containers in three replicates. Five plant species tested in this study namely, Panikum (Panicum antidotal) and napier grass (Bennisetum purpureum), squash (Cucurbita pepo), cotton (Gossypium hirsutum), sunflower (Helianthus annuus); were grown on two different polluted soil types (Mostorud Clayey soil, irrigated with contaminated water for more than 30 years and El-Gabal EL-Asfar sandyloam soil, subjected to sewage effluent irrigation for more than 50 years) in a complete randomized block experimental design to study the mobility and fate of selected heavy metals and evaluate the efficiency of the tested plant species to extract Cadmium and Zinc out of polluted soils. Data indicated that sunflower and cotton shoots accumulated the highest Cd content among the five tested plant species, Shoot concentrations of Cd were as high as 9.6 mg/kg dry matter of sunflower, followed by panikum and napier grass, cotton then squash with a range of Cd between 9.6 to 1.6 mg/kg dry matter in case of the alluvium soil. However in the sandy soil, sunflower Cd -shoots were > penakium> napier grass > cotton> Squash with a lower order of magnitude which could be explained by the lower Cd -content in sandy soil compared to the alluvial soil .Calculation of recovery percentage based on Cd and Zn removed from the soil after cultivation ranged between 5.9 to 27.4 % and 16.1 to 49.1% of total initial Cd and Zn, Respectively. However, The percentage of Cd and Zn -removed by plant shoots from the initial total varied between 27.6 to 37.5% and 25.3 and 36.8 % of the removed Cd and Zn, Respectively, whereas the lowest values were observed in case of squash for Cd and Zn. As expected plant roots exhibited higher Cd and Zn accumulation than in shoots by 2-3 folds. Sunflower roots showed the highest Cd

  19. COMPARISON OF DIFFERENT EXTRACTION METHODS REPRESENTING AVAILABLE AND TOTAL CONCENTRATIONS OF Cd, Cu, Fe, Mn and Zn IN SOIL

    Directory of Open Access Journals (Sweden)

    Vladimir Ivezić

    2013-06-01

    Full Text Available Various extraction methods are used to predict plant uptake of trace metals. Most commonly it is total concentration that is used for risk assessment and evaluation of trace metal availability. However, recent studies showed that total concentration is a poor indicator of availability while concentrations in soil solution show good correlation with plant uptake. Present study was conducted on magricultural soils with low levels of trace metals where 45 soil samples were collected from different soil types. The main objective was to compare four different extraction methods and examine how total and reactive (EDTA trace metal concentrations correlate ,with soil solution concentration (in this study determined by water extraction. The samples were analyzed by four extraction methods: strong acid extraction (ultra-pure HNO3 extraction and aqua regia, weak acid extraction by EDTA and the most available fraction, fraction in soil solution, were represented by water extraction (weakest extractant. Five elements were investigated (Cd, Cu, Fe, Mn and Zn. Water extraction significantly correlated with EDTA extraction for Cu, Fe and Mn, while total extraction (HNO3 extraction and aqua regia correlated significantly with water extraction only for Cu. No correlation between water extraction and total extraction confirmed poor role of total concentration as an indicator of availability. EDTA extraction can be used to represent reactive pool of trace metals in soil but it should be also taken with caution when using it to describe available fraction.

  20. SUPERCRITICAL FLUID EXTRACTION OF POLYCYCLIC AROMATIC HYDROCARBON MIXTURES FROM CONTAMINATED SOILS

    Science.gov (United States)

    Highly contaminated (with PAHs) topsoils were extracted with supercritical CO2 to determine the feasibility and mechanism of supercritical fluid extraction (SFE). Effect of SCF density, temperature, cosolvent type and amount, and of slurrying the soil with water were ...

  1. INVESTIGATION OF METHODS OF DNA EXTRACTION FROM PLANT ORIGIN OBJECTS AND FOODS BASED ON THEM

    Directory of Open Access Journals (Sweden)

    L. S. Dyshlyuk

    2014-01-01

    Full Text Available For the last decades modern and highly efficient methods of determining the quality and safety of food products, based on the application of the latest scientific achievements were developed in the world. A special place is given to the methods based on achievements of molecular biology and genetics. At the present stage of development in the field of assessing the quality of raw materials and processed food products much attention is given to highly accurate, sensitive and specific research methods, the method of polymerase chain reaction (PCR occupying a leading place among them. PCR is a sophisticated method that simulates the natural DNA replication and allows to detect a single specific DNA molecule in the presence of millions of other molecules. The key point in the preparation of material for PCR is the extraction of nucleic acids. The low content of DNA in plant material and the high concentration of secondary metabolites complicate the process of extraction. The key solution to this problem is highly effective method of extraction, which allows to obtain the DNA of adequate quality and purity. Comparative analysis of methods for the extraction of nucleic acids from fruit raw materials and products based on them was carried out in the study. General analysis of the experimental data allowed us to determine the most efficient method for DNA extracting. In the comparative analysis it was found out that to extract DNA from plant raw materials and food products prepared on their basis it is the most suitable to use "Sorb-GMO-A" reactants kit (set. The approach described gives us a brilliant opportunity to obtain deoxyribonucleic acid proper quality and purity.

  2. A simple Chelex protocol for DNA extraction from Anopheles spp.

    Science.gov (United States)

    Musapa, Mulenga; Kumwenda, Taida; Mkulama, Mtawa; Chishimba, Sandra; Norris, Douglas E; Thuma, Philip E; Mharakurwa, Sungano

    2013-01-09

    Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens. We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km(2) vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 μl of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 μl was retained while the other 10 μl was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol(9,10). The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction(9). Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality, a PCR for identification of Anopheles gambiae sibling species(10) and a nested PCR for typing of Plasmodium falciparum infection

  3. Toxic reagents and expensive equipment: are they really necessary for the extraction of good quality fungal DNA?

    Science.gov (United States)

    Rodrigues, P; Venâncio, A; Lima, N

    2018-01-01

    The aim of this work was to evaluate a fungal DNA extraction procedure with the lowest inputs in terms of time as well as of expensive and toxic chemicals, but able to consistently produce genomic DNA of good quality for PCR purposes. Two types of fungal biological material were tested - mycelium and conidia - combined with two protocols for DNA extraction using Sodium Dodecyl Sulphate (SDS) and Cetyl Trimethyl Ammonium Bromide as extraction buffers and glass beads for mechanical disruption of cell walls. Our results showed that conidia and SDS buffer was the combination that lead to the best DNA quality and yield, with the lowest variation between samples. This study clearly demonstrates that it is possible to obtain high yield and pure DNA from pigmented conidia without the use of strong cell disrupting procedures and of toxic reagents. There are numerous methods for DNA extraction from fungi. Some rely on expensive commercial kits and/or equipments, unavailable for many laboratories, or make use of toxic chemicals such as chloroform, phenol and mercaptoethanol. This study clearly demonstrates that it is possible to obtain high yields of pure DNA from pigmented conidia without the use of strong and expensive cell disrupting procedures and of toxic reagents. The method herein described is simultaneously inexpensive and adequate to DNA extraction from several different types of fungi. © 2017 The Society for Applied Microbiology.

  4. Comparison of three mycobacterial DNA extraction methods from extrapulmonary samples for PCR assay

    Directory of Open Access Journals (Sweden)

    Khandaker Shadia

    2012-01-01

    Full Text Available Sensitivity of the molecular diagnostic tests of extrapulmonary tuberculosis largely depends upon the efficiency of DNA extraction methods. The objective of our study was to compare three methods of extracting DNA of Mycobacterium tuberculosis for testing by polymerase chain reaction. All three methods; heating, heating with sonication and addition of lysis buffer with heating and sonication were implicated on 20 extrapulmonary samples. PCR positivity was 2 (10%, 4 (20% and 7 (35% in the samples extracted by heating, heat+sonication and heat+sonication+lysis buffer method respectively. Of the extraction methods evaluated, maximum PCR positive results were achieved by combined heat, sonication and lysis buffer method which can be applied in routine clinical practice. Ibrahim Med. Coll. J. 2012; 6(1: 9-11

  5. Identity of active methanotrophs in landfill cover soil as revealed by DNA-stable isotope probing.

    Science.gov (United States)

    Cébron, Aurélie; Bodrossy, Levente; Chen, Yin; Singer, Andrew C; Thompson, Ian P; Prosser, James I; Murrell, J Colin

    2007-10-01

    A considerable amount of methane produced during decomposition of landfill waste can be oxidized in landfill cover soil by methane-oxidizing bacteria (methanotrophs) thus reducing greenhouse gas emissions to the atmosphere. The identity of active methanotrophs in Roscommon landfill cover soil, a slightly acidic peat soil, was assessed by DNA-stable isotope probing (SIP). Landfill cover soil slurries were incubated with (13)C-labelled methane and under either nutrient-rich nitrate mineral salt medium or water. The identity of active methanotrophs was revealed by analysis of (13)C-labelled DNA fractions. The diversity of functional genes (pmoA and mmoX) and 16S rRNA genes was analyzed using clone libraries, microarrays and denaturing gradient gel electrophoresis. 16S rRNA gene analysis revealed that the cover soil was mainly dominated by Type II methanotrophs closely related to the genera Methylocella and Methylocapsa and to Methylocystis species. These results were supported by analysis of mmoX genes in (13)C-DNA. Analysis of pmoA gene diversity indicated that a significant proportion of active bacteria were also closely related to the Type I methanotrophs, Methylobacter and Methylomonas species. Environmental conditions in the slightly acidic peat soil from Roscommon landfill cover allow establishment of both Type I and Type II methanotrophs.

  6. Role of Various Extractants in Removing Group-IIB Elements of Soils Incubated with EDTA

    Directory of Open Access Journals (Sweden)

    Tanmoy Karak

    2004-01-01

    Full Text Available This paper presents the results of an experimental investigation undertaken to evaluate different extractant solutions viz. HCl, Mg(NO32, and DTPA with the range of concentration from 0.001 to 0.1N after incubation with group-IIB metals (Zn, Cd, and Hg and EDTA to understand the capability to remove Zn, Cd, and Hg from soils. Two noncontaminated soils, one acidic (GHL and the other alkaline (KAP, in reaction were taken from an agricultural field of West Bengal, India for this investigation. Experiments were conducted on these two soils spiked with ZnII, CdII, and HgII in concentrations of 612, 321, and 215 mg/kg for soil GHL and 778, 298, and 157 mg/kg for soil KAP, respectively, which simulate typical electroplating waste contamination. The removal of Zn, Cd, and Hg in soil GHL within the range of HCl concentrations was 8.2–16.5, 12.2–19.1, and 4.3–6.9 whereas these were 6.5–7.6, 8.5–14.1, and 3.2–5.2 in soil KAP. The removal of Zn, Cd, and Hg in soil GHL within the range of Mg(NO32 concentrations were 12.2–28.5, 19.1–24.6, and 18.2–19.1 whereas these were 9.1–12.1, 8.3–12.1, and 10.6–48.1 in soil KAP. For DTPA extractant, the percent removal of metal was found to be significantly higher than the other two extractants, which corroborates that DTPA is a better extractant for soil cleaning.

  7. Improved Methods of Carnivore Faecal Sample Preservation, DNA Extraction and Quantification for Accurate Genotyping of Wild Tigers

    Science.gov (United States)

    Harika, Katakam; Mahla, Ranjeet Singh; Shivaji, Sisinthy

    2012-01-01

    Background Non-invasively collected samples allow a variety of genetic studies on endangered and elusive species. However due to low amplification success and high genotyping error rates fewer samples can be identified up to the individual level. Number of PCRs needed to obtain reliable genotypes also noticeably increase. Methods We developed a quantitative PCR assay to measure and grade amplifiable nuclear DNA in feline faecal extracts. We determined DNA degradation in experimentally aged faecal samples and tested a suite of pre-PCR protocols to considerably improve DNA retrieval. Results Average DNA concentrations of Grade I, II and III extracts were 982pg/µl, 9.5pg/µl and 0.4pg/µl respectively. Nearly 10% of extracts had no amplifiable DNA. Microsatellite PCR success and allelic dropout rates were 92% and 1.5% in Grade I, 79% and 5% in Grade II, and 54% and 16% in Grade III respectively. Our results on experimentally aged faecal samples showed that ageing has a significant effect on quantity and quality of amplifiable DNA (pDNA degradation occurs within 3 days of exposure to direct sunlight. DNA concentrations of Day 1 samples stored by ethanol and silica methods for a month varied significantly from fresh Day 1 extracts (p0.05). DNA concentrations of fresh tiger and leopard faecal extracts without addition of carrier RNA were 816.5pg/µl (±115.5) and 690.1pg/µl (±207.1), while concentrations with addition of carrier RNA were 49414.5pg/µl (±9370.6) and 20982.7pg/µl (±6835.8) respectively. Conclusions Our results indicate that carnivore faecal samples should be collected as freshly as possible, are better preserved by two-step method and should be extracted with addition of carrier RNA. We recommend quantification of template DNA as this facilitates several downstream protocols. PMID:23071624

  8. Ancient DNA in historical parchments - identifying a procedure for extraction and amplification of genetic material.

    Science.gov (United States)

    Lech, T

    2016-05-06

    Historical parchments in the form of documents, manuscripts, books, or letters, make up a large portion of cultural heritage collections. Their priceless historical value is associated with not only their content, but also the information hidden in the DNA deposited on them. Analyses of ancient DNA (aDNA) retrieved from parchments can be used in various investigations, including, but not limited to, studying their authentication, tracing the development of the culture, diplomacy, and technology, as well as obtaining information on the usage and domestication of animals. This article proposes and verifies a procedure for aDNA recovery from historical parchments and its appropriate preparation for further analyses. This study involved experimental selection of an aDNA extraction method with the highest efficiency and quality of extracted genetic material, from among the multi-stage phenol-chloroform extraction methods, and the modern, column-based techniques that use selective DNA-binding membranes. Moreover, current techniques to amplify entire genetic material were questioned, and the possibility of using mitochondrial DNA for species identification was analyzed. The usefulness of the proposed procedure was successfully confirmed in identification tests of historical parchments dating back to the 13-16th century AD.

  9. Development of metabarcoding for tracking changes of soil fauna community under stress by application of ash

    DEFF Research Database (Denmark)

    Qin, J; de Groot, G.A.; Hansen, L. H.

    Ash is a waste product from combustion of bio-fuel in power plants. Application of ash on soil ensures nutrient recycling, but detrimental ecotoxicological consequences may arise since ash is a complex mixture that may contain compounds affecting soil invertebrates and their food and habitat...... and species. DNA metabarcoding, which couples the principle of DNA barcoding with next generation sequencing technology, has the potential to simplify community diversity monitoring. However, sampling and DNA extraction methods for the purpose of soil microarthropod metabarcoding have not been yet fully...

  10. Differential Nuclear and Mitochondrial DNA Preservation in Post-Mortem Teeth with Implications for Forensic and Ancient DNA Studies

    Science.gov (United States)

    Higgins, Denice; Rohrlach, Adam B.; Kaidonis, John; Townsend, Grant; Austin, Jeremy J.

    2015-01-01

    Major advances in genetic analysis of skeletal remains have been made over the last decade, primarily due to improvements in post-DNA-extraction techniques. Despite this, a key challenge for DNA analysis of skeletal remains is the limited yield of DNA recovered from these poorly preserved samples. Enhanced DNA recovery by improved sampling and extraction techniques would allow further advancements. However, little is known about the post-mortem kinetics of DNA degradation and whether the rate of degradation varies between nuclear and mitochondrial DNA or across different skeletal tissues. This knowledge, along with information regarding ante-mortem DNA distribution within skeletal elements, would inform sampling protocols facilitating development of improved extraction processes. Here we present a combined genetic and histological examination of DNA content and rates of DNA degradation in the different tooth tissues of 150 human molars over short-medium post-mortem intervals. DNA was extracted from coronal dentine, root dentine, cementum and pulp of 114 teeth via a silica column method and the remaining 36 teeth were examined histologically. Real time quantification assays based on two nuclear DNA fragments (67 bp and 156 bp) and one mitochondrial DNA fragment (77 bp) showed nuclear and mitochondrial DNA degraded exponentially, but at different rates, depending on post-mortem interval and soil temperature. In contrast to previous studies, we identified differential survival of nuclear and mtDNA in different tooth tissues. Futhermore histological examination showed pulp and dentine were rapidly affected by loss of structural integrity, and pulp was completely destroyed in a relatively short time period. Conversely, cementum showed little structural change over the same time period. Finally, we confirm that targeted sampling of cementum from teeth buried for up to 16 months can provide a reliable source of nuclear DNA for STR-based genotyping using standard

  11. Direct human DNA protection by Coriolus versicolor (Yunzhi) extract.

    Science.gov (United States)

    Szeto, Yim Tong; Lau, Po Chun; Kalle, Wouter; Pak, Sok Cheon

    2013-07-01

    Scientific evidence has shown Coriolus versicolor (L. ex Fr.) Quel (also known as Yunzhi) has the role of immunomodulator in therapeutic effect. The aim of this in vitro study was to investigate the antioxidative effect of Yunzhi and to explore the mechanisms behind its DNA protection. Commercial Yunzhi extract was dissolved in water and diluted in five concentrations (10(1)-10(5) μg/L) with appropriate buffers. Lymphocytes harvested from three healthy subjects were incubated with Yunzhi extract for 30 min. Cells were then subjected to 5 min oxidant challenge by 45 μM hydrogen peroxide. The standard alkaline comet (SAC) assay and lysed cell comet (LCC) assay were performed in parallel. DNA damage of each treatment was scored under a fluorescence microscope and compared with the cells without Yunzhi pretreatment. U-shaped dose-response was seen in both versions of the comet assay. Yunzhi at 10(4) μg/L demonstrated a genoprotective effect against oxidative damage in the SAC assay (25% decrease in comet score). In the LCC assay, a trend of protection in lymphocytes was observed but it did not reach statistical significance. A direct antioxidant effect of Yunzhi against oxidant challenge on the DNA of lymphocytes was evidenced. The active component in Yunzhi was likely to be membrane permeable.

  12. Rapid extraction of PCDD/Fs from soil and fly ash samples. Pressurized fluid extraction (PFE) and microwave-assisted extraction (MAE)

    Energy Technology Data Exchange (ETDEWEB)

    Sanz, P.; Fabrellas, B. [Centro de Investigaciones Energeticas Medioambientales y Tecnologicas (CIEMAT), Madrid (Spain)

    2004-09-15

    The main reference extraction method in the analysis of polychlorinated dibenzop- dioxins and dibenzofurans (PCDD/Fs) is still the Soxhlet extraction. But it requires long extraction times (up to 24 hs), large volumes of hazardous organic solvents (100-300 ml) and its automation is limited. Pressurized Fluid Extraction (PFE) and Microwave-Assisted Extraction (MAE) are two relatively new extraction techniques that reduce the time and the volume of solvent required for extraction. However, very different PFE extraction conditions are found for the same enviromental matrices in the literature. MAE is not a extraction technique very applied for the analysis of PCDD/Fs yet, although it is used for the determination of other organic compounds, such as PCBs and PAHs. In this study, PFE and MAE extraction conditions were optimized to determine PCDDs y PCDFs in fly ash and soil/sediment samples. Conventional Soxhlet extraction with toluene was used to compare the extraction efficiency of both techniques.

  13. Extraction of PCR-amplifiable genomic DNA from Bacillus anthracisspores

    Energy Technology Data Exchange (ETDEWEB)

    Torok, Tamas

    2003-05-19

    Bacterial endospore disruption and nucleic acid extractionresulting in DNA of PCR-amplifiable quality and quantity are not trivial.Responding to the needs of the Hazardous Materials Response Unit (HMRU),Laboratory Division, Federal Bureau of Investigation, protocols weredeveloped to close these gaps. Effectiveness and reproducibility of thetechniques were validated with laboratory grown pure spores of Bacillusanthracis and its close phylogenetic neighbors, and with spiked soils anddamaged samples.

  14. Automated extraction of DNA from biological stains on fabric from crime cases. A comparison of a manual and three automated methods.

    Science.gov (United States)

    Stangegaard, Michael; Hjort, Benjamin B; Hansen, Thomas N; Hoflund, Anders; Mogensen, Helle S; Hansen, Anders J; Morling, Niels

    2013-05-01

    The presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. DNA extraction from fabric for forensic genetic purposes may be challenging due to the occasional presence of PCR inhibitors that may be co-extracted with the DNA. Using 120 forensic trace evidence samples consisting of various types of fabric, we compared three automated DNA extraction methods based on magnetic beads (PrepFiler Express Forensic DNA Extraction Kit on an AutoMate Express, QIAsyphony DNA Investigator kit either with the sample pre-treatment recommended by Qiagen or an in-house optimized sample pre-treatment on a QIAsymphony SP) and one manual method (Chelex) with the aim of reducing the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable STR-profiles. A total of 480 samples were processed. The highest DNA recovery was obtained with the PrepFiler Express kit on an AutoMate Express while the lowest DNA recovery was obtained using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen. Extraction using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen resulted in the lowest percentage of PCR inhibition (0%) while extraction using manual Chelex resulted in the highest percentage of PCR inhibition (51%). The largest number of reportable STR-profiles was obtained with DNA from samples extracted with the PrepFiler Express kit (75%) while the lowest number was obtained with DNA from samples extracted using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen (41%). Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  15. Influence of plants on the chemical extractability and biodegradability of 2,4-dichlorophenol in soil

    International Nuclear Information System (INIS)

    Boucard, Tatiana K.; Bardgett, Richard D.; Jones, Kevin C.; Semple, Kirk

    2005-01-01

    This study investigated the fate and behaviour of [UL- 14 C] 2,4-dichlorophenol (DCP) in planted (Lolium perenne L.) and unplanted soils over 57 days. Extractability of [UL- 14 C] 2,4-DCP associated activity was measured using calcium chloride (CaCl 2 ), acetonitrile-water and dichloromethane (DCM) extractions. Biodegradability of [UL- 14 C] 2,4-DCP associated activity was assessed through measurement of 14 CO 2 production by a degrader inoculum (Burkholderia sp.). Although extractability and mineralisation of [UL- 14 C] 2,4-DCP associated activity decreased significantly in both planted and unplanted soils, plants appeared to enhance the sequestration process. After 57 days, in unplanted soil, 27% of the remaining [UL- 14 C] 2,4-DCP associated activity was mineralised by Burkholderia sp., and 13%, 48%, and 38% of 14 C-activity were extracted by CaCl 2 , acetonitrile-water and DCM, respectively. However, after 57 days, in planted soils, only 10% of the [UL- 14 C] 2,4-DCP associated activity was available for mineralisation, whilst extractability was reduced to 2% by CaCl 2 , 17% by acetonitrile-water and 11% by DCM. This may be due to the effect of plants on soil moisture conditions, which leads to modification of the soil structure and trapping of the compound. However, the influence of plants on soil biological and chemical properties may also play a role in the ageing process

  16. Remediation of heavy-metal-contaminated soil using chelant extraction: Feasibility studies

    Energy Technology Data Exchange (ETDEWEB)

    Peters, R.W.; Miller, G.; Taylor, J.D.; Schneider, J.F.; Zellmer, S.; Edgar, D.E.; Johnson, D.O.

    1993-08-01

    Results are presented of a laboratory investigation conducted to determine the efficacy of using chelating agents to extract heavy metals (Pb, Cd, Cr, Ba, Cu, and Zn) from soil, the primary focus being on the extraction of lead from the soil. Results from the batch-shaker studies and emphasizes the columnar extraction studies are described. The chelating agents studied included ethylenediaminetetraacetic acid (EDTA) and citric acid, in addition to water. Concentrations of the chelants ranged from 0.01 to 0.05 M; the suspension pH was varied between 3 and 8. Results showed that the removal of lead using citric acid and water was somewhat pH-dependent. For the batch-shaker studies, the results indicated that EDTA was more effective at removing Cd, Cu, Pb, and Zn than was citric acid (both present at 0.01 M). EDTA and citric acid were equally effective in mobilizing Cr and Ba from the soil. Heavy metals removal was slightly more effective in the more acidic region (pH {le} 5).

  17. System of extraction of volatiles from soil using microwave processes

    Science.gov (United States)

    Ethridge, Edwin C. (Inventor); Kaukler, William F. (Inventor)

    2013-01-01

    A device for the extraction and collection of volatiles from soil or planetary regolith. The device utilizes core drilled holes to gain access to underlying volatiles below the surface. Microwave energy beamed into the holes penetrates through the soil or regolith to heat it, and thereby produces vapor by sublimation. The device confines and transports volatiles to a cold trap for collection.

  18. DNA is a co-factor for its own replication in Xenopus egg extracts

    NARCIS (Netherlands)

    Lebofsky, Ronald; van Oijen, Antoine M.; Walter, Johannes C.

    Soluble Xenopus egg extracts efficiently replicate added plasmids using a physiological mechanism, and thus represent a powerful system to understand vertebrate DNA replication. Surprisingly, DNA replication in this system is highly sensitive to plasmid concentration, being undetectable below

  19. Phyto extraction of 99Tc on soil cores with aged contamination

    International Nuclear Information System (INIS)

    Massoura, S.T.; Echevarria, G.; Morel, J.L.; Massoura, S.T.; Leclerc-Cessac, E.; Denys, D.

    2004-01-01

    99 Tc is an artificial radionuclide which is found in high-activity and long-lived nuclear waste. This work was designed to study the phyto-extraction of 99 Tc in soils that had received aged contamination and to monitor the resulting 99 Tc concentrations in the soil solution of undisturbed soil cores in a greenhouse. Undisturbed soil cores had been sampled previously from a Rendzic Leptosol (R), a Fluvic cambisol (F) and a Dystric cambisol (D), using 0.5-m diameter PVC tubing (3 samples/soil type) without disturbing soil structure (1). Each core was equipped with two nylon porous cups (respectively 20 and 35 cm deep) and a final leachate collector. A 99 TcNO 3 solution had been supplied at the soil surface of each core during the two previous years (4200 kBq in total) in which maize and wheat had been successively cropped. These two crops had already removed 30-65% of total contamination before the present study. After the second year no more 99 Tc was added to the cores. Thereafter, Lolium perenne was cultivated for 20 successive months. 99 Tc was determined in both plant aerial parts and water samples (from both cups and collectors), and the balance of 99 Tc in the system was established after phyto-extraction. Results showed that transfer of 99 Tc to plants vary among soils: 7% on soil R to 11% on soil D. Concentration of 99 Tc in the porous cups dramatically decreased in all soils. The plants maintained low and stable concentration levels of 99 Tc in the soil solution which decreased the potential migration of the radionuclide through the cores: The leaching of 99 Tc in the final collectors of the R soil cores decreased from 18 to 1.7 Bq mL -1 . (author)

  20. Multiresidual determination of pesticides in agricultural soil sample using Quechers extraction methodology

    International Nuclear Information System (INIS)

    Castro Garcia, Consuelo del Pilar

    2011-01-01

    To achieve a sustainable agricultural production there are used different organic and inorganic products, among them we found the fertilizers and pesticides. When they are applied most of the product falls to the ground, generating significant sources of pollution in the areas near the application and depending on the mobility of the pesticide, it can reach more remote areas. That is why it is important to determine the pesticide residues in soil after their application, being the selection of the extraction method crucial for the subsequent traces detection. In the present work there was evaluated the QUECHERS extraction technique, a method used in food but modified for a different and complex matrix like soil in order to achieve acceptable efficiencies multi-residue extraction of 20 pesticides and their subsequent determination by gas chromatography with electron capture and mass detection. The method was applied for the determination of pesticides in three soil samples from an agricultural site with different slopes between them. The Results indicated that 75% of the pesticides tested had acceptable efficiencies, thus meeting the objective of achieving multiresidue determination of pesticides in agricultural soil samples by extraction methodology QUECHERS. Besides, the presence of the fungicide penconazole was only detected in the three samples, being the highest concentration of pesticide found in the area with less slope (V_A_B_A_J_O) (author)

  1. [Aging Law of PAHs in Contaminated Soil and Their Enrichment in Earthworms Characterized by Chemical Extraction Techniques].

    Science.gov (United States)

    Zhang, Ya-nan; Yang, Xing-lun; Bian, Yong-rong; Gu, Cheng-gang; Liu, Zong-tang; Li, Jiao; Wang, Dai-zhang; Jiang, Xin

    2015-12-01

    To evaluate the effect of aging on the availability of PAHs, chemical extraction by exhaustive ( ASE extraction) and nonexhaustive techniques (Tenax-TA extraction, hydroxypropyl-p-cyclodextrin ( HPCD ) extraction, n-butyl alcohol ( BuOH) extraction) as well as PAHs accumulation in earthworms (Eisenia fetida) were conducted in yellow soil from Baguazhou, Nanjing, China, and red soil from Hainan, China, spiked with phenanthrene, pryene and benzo(a) pyrene and aged 0, 7, 15, 30 and 60 days. The results showed that the concentration of PAHs extracted by ASE and three nonexhaustive techniques and accumulated by earthworms significantly decreased with aging time, except the ASE extracted concentration between 30-and 60-day aging time. Furthermore, the relationships were studied in this experiment between chemical extracted PAHs concentration and accumulated concentration in earthworms. PAHs accumulated concentration in earthworms was not significantly correlated with the exhaustive extracted concentration of PAHs in soil (R² 0.44-0.56), which indicated that ASE extraction techniques could not predict PAHs bioavailability to earthworms because it overestimated the risk of PAHs. However, the PAHs accumulated concentration in earthworms was significantly correlated with the three nonexhaustive extracted concentrations of PAHs in soil, which indicated that all the three nonexhaustive techniques could predict PAHs bioavailability to earthworm to some extent, among which, HPCD extraction (R² 0.94-0.99) was better than Tenax-TA extraction (R² 0.62-0.87) and BuOH extraction (R² 0.69-0.94). So HPCD extraction was a more appropriate and reliable technique to predict bioavailability of PAHs in soil.

  2. CGH and SNP array using DNA extracted from fixed cytogenetic preparations and long-term refrigerated bone marrow specimens

    Directory of Open Access Journals (Sweden)

    MacKinnon Ruth N

    2012-02-01

    Full Text Available Abstract Background The analysis of nucleic acids is limited by the availability of archival specimens and the quality and amount of the extracted material. Archived cytogenetic preparations are stored in many laboratories and are a potential source of total genomic DNA for array karyotyping and other applications. Array CGH using DNA from fixed cytogenetic preparations has been described, but it is not known whether it can be used for SNP arrays. Diagnostic bone marrow specimens taken during the assessment of hematological malignancies are also a potential source of DNA, but it is generally assumed that DNA must be extracted, or the specimen frozen, within a day or two of collection, to obtain DNA suitable for further analysis. We have assessed DNA extracted from these materials for both SNP array and array CGH. Results We show that both SNP array and array CGH can be performed on genomic DNA extracted from cytogenetic specimens stored in Carnoy's fixative, and from bone marrow which has been stored unfrozen, at 4°C, for at least 36 days. We describe a procedure for extracting a usable concentration of total genomic DNA from cytogenetic suspensions of low cellularity. Conclusions The ability to use these archival specimens for DNA-based analysis increases the potential for retrospective genetic analysis of clinical specimens. Fixed cytogenetic preparations and long-term refrigerated bone marrow both provide DNA suitable for array karyotyping, and may be suitable for a wider range of analytical procedures.

  3. Optimizing factors influencing DNA extraction from fresh whole avian ...

    African Journals Online (AJOL)

    A study was conducted to optimize the efficient combination of lysis buffer, proteinase K, incubation time, phenol-chloroform-isoamyl alcohol (PCI) volume, spinning rate (rpm), and precipitation agent on quantity and quality of DNA extracted from various volumes of avian blood. Blood samples were collected in EDTA and ...

  4. The Effect of a Grape Seed Extract on Radiation-Induced DNA Damage in Human Lymphocytes

    Science.gov (United States)

    Dicu, Tiberius; Postescu, Ion D.; Foriş, Vasile; Brie, Ioana; Fischer-Fodor, Eva; Cernea, Valentin; Moldovan, Mircea; Cosma, Constantin

    2009-05-01

    Plant-derived antioxidants due to their phenolic compounds content are reported as potential candidates for reducing the levels of oxidative stress in living organisms. Grape seed extracts are very potent antioxidants and exhibit numerous interesting pharmacologic activities. Hydroethanolic (50/50, v/v) standardized extract was obtained from red grape seed (Vitis vinifera, variety Burgund Mare—BM). The total polyphenols content was evaluated by Folin-Ciocalteu procedure and expressed as μEq Gallic Acid/ml. The aim of this study was to evaluate the potential antioxidant effects of different concentrations of BM extract against 60Co γ-rays induced DNA damage in human lymphocytes. Samples of human lymphocytes were incubated with BM extract (12.5, 25.0 and 37.5 μEq GA/ml, respectively) administered at 30 minutes before in vitro irradiation with γ-rays (2 Gy). The DNA damage and repair in lymphocytes were evaluated using alkaline comet assay. Using the lesion score, the radiation-induced DNA damage was found to be significantly different (pextract (except the lymphocytes treated with 37.5 μEq GA/ml BM extract). DNA repair analyzed by incubating the irradiated cells at 37° C and 5% CO2 atmosphere for 2 h, indicated a significant difference (pextract, immediately and two hours after irradiation. These results suggest radioprotective effects after treatment with BM extract in human lymphocytes.

  5. Evaluation of dna extraction methods of the Salmonella sp. bacterium in artificially infected chickens eggs

    Directory of Open Access Journals (Sweden)

    Ana Cristina dos Reis Ferreira

    2015-06-01

    Full Text Available ABSTRACT. Ferreira A.C.dosR. & dos Santos B.M. [Evaluation of dna extraction methods of the Salmonella sp. bacterium in artificially infected chickens eggs.] Avaliação de três métodos de extração de DNA de Salmonella sp. em ovos de galinhas contaminados artificialmente. Revista Brasileira de Medicina Veterinária, 37(2:115-119, 2015. Departamento de Veterinária, Universidade Federal de Viçosa, Campus Universitário, Av. Peter Henry Rolfs, s/n, Viçosa, MG 36571-000, Brasil. E-mail: bmsantos@ufv.br The present study evaluated the efficiency of different protocols for the genomic DNA extraction of Salmonella bacteria in chicken eggs free of specific pathogens – SPF. Seventy-five eggs were used and divided into five groups with fifteen eggs each. Three of the five groups of eggs were inoculated with enteric Salmonella cultures. One of the five groups was inoculated with Escherichia coli bacterium culture. And another group of eggs was the negative control that received saline solution 0.85% infertile. The eggs were incubated on a temperature that varied from 20 to 25°C during 24, 48 and 72 hours. Five yolks of each group were collected every 24 hours. These yolks were homogenized and centrifuged during 10 minutes. The supernatant was rejected. After the discard, PBS ph 7.2 was added and centrifuged again. The sediment obtained of each group was used for the extraction of bacterial genomic DNA. Silica particles and a commercial kit were utilized as the extraction methods. The extracted DNA was kept on a temperature of 20°C until the evaluation through PCR. The primers utilized were related with the invA gene and they were the following: 5’ GTA AAA TTA TCG CCA CGT TCG GGC AA 3’ and 5’ TCA TCG CAC CGT CAA AGG AAC C 3’. The amplification products were visualized in transilluminator with ultraviolet light. The obtained results through the bacterial DNA extractions demonstrated that the extraction method utilizing silica particles was

  6. Alkaline extraction: can it be used for the removal of 137Cs from soil?

    International Nuclear Information System (INIS)

    Nageldinger, G.; Flowers, A.; Entwistle, J.

    1998-01-01

    A titration extraction procedure was applied to a silty clay which was spiked with 137 Cs. At least 20% of spiked 137 Cs silty clay was found to be associated with the humic acid (HA) fraction. This shows that HA contributes significantly to cesium fixing in soil and cesium is not only fixed between regular ion exchange complexes (REC) and specific sites in the frayed edges of clay minerals (FES). About 85% of 137 Cs was found in the 12M HCl extract. The titration extraction procedure was found useful for investigation of the impact of the soil pH on the radiocesium mobility. Only trace amounts of cesium were found between pH 1 to 7, demonstrating a high immobility of cesium over this pH range. If the alkaline digestion is applied to soil for the removal of radiocesium, then these can be significantly separated from the extract by protonation of the extracted HA. (P.A.)

  7. Modifier free supercritical fluid extraction of uranium from sintered UO2, soil and ore samples

    International Nuclear Information System (INIS)

    Kanekar, A.S.; Pathak, P.N.; Acharya, R.; Mohapatra, P.K.; Manchanda, V.K.

    2011-01-01

    Direct extraction of uranium from different samples viz. sintered UO 2 , soil and ores was carried out by modifier free supercritical fluid using tri-n-butyl phosphate-nitric acid (TBP-HNO 3 ) adduct as extractant. These studies showed that pre-equilibration with more concentrated nitric acid helps in better dissolution and extraction of uranium from sintered UO 2 samples. Modifier free supercritical fluid extraction appears attractive with respect to minimization of secondary wastes. This method resulted 80-100% extraction of uranium from different soil/ore samples. The results were confirmed by performing neutron activation analysis of original (before extraction) and residue (after extraction) samples. (author)

  8. Optimization of conditions to extract high quality DNA for PCR analysis from whole blood using SDS-proteinase K method

    Directory of Open Access Journals (Sweden)

    Wajhul Qamar

    2017-11-01

    Full Text Available In case of studies associated with human genetics, genomics, and pharmacogenetics the genomic DNA is extracted from the buccal cells, whole blood etc. Several methods are exploited by the researchers to extract DNA from the whole blood. One of these methods, which utilizes cell lysis and proteolytic properties of sodium dodecyl sulfate (SDS and proteinase K respectively, might also be called SDS-PK method. It does not include any hazardous chemicals such as phenol or chloroform and is inexpensive. However, several researchers report the same method with different formulas and conditions. During our experiments with whole blood DNA extraction we experienced problems such as protein contamination, DNA purity and yield when followed some SDS-PK protocols reported elsewhere. A260/A280 and A260/A230 ratios along with PCR amplification give a clear idea about the procedure that was followed to extract the DNA. In an effort to increase the DNA purity from human whole blood, we pointed out some steps of the protocol that play a crucial role in determining the extraction of high quality DNA.

  9. DNA barcodes for soil animal taxonomy Código de barras de DNA para a taxonomia de animais do solo

    Directory of Open Access Journals (Sweden)

    Rodolphe Rougerie

    2009-08-01

    Full Text Available The biodiversity of soil communities remains very poorly known and understood. Soil biological sciences are strongly affected by the taxonomic crisis, and most groups of animals in that biota suffer from a strong taxonomic impediment. The objective of this work was to investigate how DNA barcoding - a novel method using a microgenomic tag for species identification and discrimination - permits better evaluation of the taxonomy of soil biota. A total of 1,152 barcode sequences were analyzed for two major groups of animals, collembolans and earthworms, which presented broad taxonomic and geographic sampling. Besides strongly reflecting the taxonomic impediment for both groups, with a large number of species-level divergent lineages remaining unnamed so far, the results also highlight a high level (15% of cryptic diversity within known species of both earthworms and collembolans. These results are supportive of recent local studies using a similar approach. Within an impeded taxonomic system for soil animals, DNA-assisted identification tools can facilitate and improve biodiversity exploration and description. DNA-barcoding campaigns are rapidly developing in soil animals and the community of soil biologists is urged to embrace these methods.A biodiversidade das comunidades do solo continua muito pouco conhecida e entendida. A biologia do solo é fortemente afetada pela crise taxonômica, e a maior parte dos grupos de animais dessa biota sofre forte impedimento taxonômico. O objetivo deste trabalho foi determinar como o código de barras de DNA - um método novo que usa uma etiqueta microgenômica para identificação e discriminação de espécies - permite uma melhor avaliação da taxonomia da biota edáfica. Foram analisadas 1.152 sequências de códigos de barras de dois grupos principais de animais, colêmbolos e minhocas, que apresentaram ampla amostragem taxonômica e geográfica. Além de refletir fortemente o impedimento taxonômico de

  10. A simple and cost-effective method of DNA extraction from small formalin-fixed paraffin-embedded tissue for molecular oncologic testing.

    Science.gov (United States)

    Snow, Anthony N; Stence, Aaron A; Pruessner, Jonathan A; Bossler, Aaron D; Ma, Deqin

    2014-01-01

    Extraction of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing. As molecular oncology testing becomes more important for prognostic and therapeutic decision making and tissue specimens become smaller due to earlier detection of suspicious lesions and the use of fine needle aspiration methods for tissue collection, it becomes more challenging for the typical molecular pathology laboratory to obtain reliable test results. We developed a DNA extraction method to obtain sufficient quantity and high quality genomic DNA from limited FFPE tissue for molecular oncology testing using a combination of H&E stained slides, a matrix capture method and the Qiagen DNA column. THREE DNA EXTRACTION METHODS WERE COMPARED: our standard procedure of manually scraping tissue from unstained slides followed by DNA extraction using the QIAamp FFPE column (Qiagen, Valencia, CA), a glue capture method (Pinpoint Solution, Zymo Research Corp, Inc) on H&E stained slides followed by DNA extraction using either the QIAamp column or the column included with the Pinpoint kit (Zymo Research). The DNA extraction protocol was optimized. Statistical analysis was performed using the paired two-sample student's t-test. The combination of the matrix capture method with the QIAamp column gave an equivalent amount of DNA as our standard extraction method using the unstained slides and a 4.6-fold higher DNA yield than using the Zymo column included in the Pinpoint Slide Solution kit. Several molecular tests were performed and DNA purified using the new method gave the same results as for the previous methods. Using H&E stained slides allows visual confirmation of tumor cells during microdissection. The Pinpoint solution made removal of specific tissue from the slides easier and reduced the risk of contamination and tissue loss. This DNA extraction method is simple, cost-effective, and blends with our current workflow requiring no additional equipment.

  11. Effect of aluminum, zinc, copper, and lead on the acid-base properties of water extracts from soils

    Science.gov (United States)

    Motuzova, G. V.; Makarychev, I. P.; Petrov, M. I.

    2013-01-01

    The potentiometric titration of water extracts from the upper horizons of taiga-zone soils by salt solutions of heavy metals (Pb, Cu, and Zn) showed that their addition is an additional source of the extract acidity because of the involvement of the metal ions in complexation with water-soluble organic substances (WSOSs). At the addition of 0.01 M water solutions of Al(NO3)3 to water extracts from soils, Al3+ ions are also involved in complexes with WSOSs, which is accompanied by stronger acidification of the extracts from the upper horizon of soddy soils (with a near-neutral reaction) than from the litter of bog-podzolic soil (with a strongly acid reaction). The effect of the Al3+ hydrolysis on the acidity of the extracts is insignificantly low in both cases. A quantitative relationship was revealed between the release of protons and the ratio of free Cu2+ ions to those complexed with WSOSs at the titration of water extracts from soils by a solution of copper salt.

  12. Novel extraction strategy of ribosomal RNA and genomic DNA from cheese for PCR-based investigations.

    Science.gov (United States)

    Bonaïti, Catherine; Parayre, Sandrine; Irlinger, Françoise

    2006-03-15

    Cheese microorganisms, such as bacteria and fungi, constitute a complex ecosystem that plays a central role in cheeses ripening. The molecular study of cheese microbial diversity and activity is essential but the extraction of high quality nucleic acid may be problematic: the cheese samples are characterised by a strong buffering capacity which negatively influenced the yield of the extracted rRNA. The objective of this study is to develop an effective method for the direct and simultaneous isolation of yeast and bacterial ribosomal RNA and genomic DNA from the same cheese samples. DNA isolation was based on a protocol used for nucleic acids isolation from anaerobic digestor, without preliminary washing step with the combined use of the action of chaotropic agent (acid guanidinium thiocyanate), detergents (SDS, N-lauroylsarcosine), chelating agent (EDTA) and a mechanical method (bead beating system). The DNA purification was carried out by two washing steps of phenol-chloroform. RNA was isolated successfully after the second acid extraction step by recovering it from the phenolic phase of the first acid extraction. The novel method yielded pure preparation of undegraded RNA accessible for reverse transcription-PCR. The extraction protocol of genomic DNA and rRNA was applicable to complex ecosystem of different cheese matrices.

  13. Aqueous CO2 vs. aqueous extraction of soils as a preparative procedure for acute toxicity testing

    International Nuclear Information System (INIS)

    Yates, G.W.; Burks, S.L.

    1994-01-01

    This study was to determine if contaminated soils extracted with supercritical CO 2 (SFE) would yield different results from soils extracted with an aqueous media. Soil samples from an abandoned oil refinery were subjected to aqueous and SFE extraction. Uncontaminated control sites were compared with contaminated sites. Each extract was analyzed for 48 hour acute Ceriodaphnia LC50s and Microtox reg-sign EC50s. Comparisons were then made between the aqueous extracts and the SFE extracts. An additional study was made with HPLC chromatographs of the SFE contaminated site extracts to determine if there was a correlation between LC50 results and peak area of different sections of the chromatograph. The 48 hour Ceriodaphnia LC50 of one contaminated site showed a significant increase in toxicity with the supercritical extract compared to the aqueous extract. All contaminated sites gave toxic responses with the supercritical procedure. The Microtox reg-sign assay showed a toxic response with 2 of the 3 contaminated sites for both aqueous and SFE extracts. Results indicate that the Ceriodaphnia assays were more sensitive than Microtox reg-sign to contaminants found in the refinery soil. SFE controls did not show adverse effects with the Ceriodaphnia, but did have a slight effect with Microtox reg-sign. The best correlation (r 2 > 0.90) between the Ceriodaphnia LC50s and the peak areas of the chromatographs was obtained for sections with an estimated log K ow of 1 to 5. SFE extraction provided a fast, efficient and inexpensive method of collecting and testing moderately non-polar to strongly non-polar organic contaminants from contaminated soils

  14. DNA Extraction by Isotachophoresis in a Microfluidic Channel

    Energy Technology Data Exchange (ETDEWEB)

    Stephenson, S J

    2011-08-10

    Biological assays have many applications. For example, forensics personnel and medical professionals use these tests to diagnose diseases and track their progression or identify pathogens and the host response to them. One limitation of these tests, however, is that most of them target only one piece of the sample - such as bacterial DNA - and other components (e.g. host genomic DNA) get in the way, even though they may be useful for different tests. To address this problem, it would be useful to extract several different substances from a complex biological sample - such as blood - in an inexpensive and efficient manner. This summer, I worked with Maxim Shusteff at Lawrence Livermore National Lab on the Rapid Automated Sample Prep project. The goal of the project is to solve the aforementioned problem by creating a system that uses a series of different extraction methods to extract cells, bacteria, and DNA from a complex biological sample. Biological assays can then be run on purified output samples. In this device, an operator could input a complex sample such as blood or saliva, and would receive separate outputs of cells, bacteria, viruses, and DNA. I had the opportunity to work this summer with isotachophoresis (ITP), a technique that can be used to extract nucleic acids from a sample. This technique is intended to be the last stage of the purification device. Isotachophoresis separates particles based on different electrophoretic mobilities. This technique is convenient for out application because free solution DNA mobility is approximately equal for DNA longer than 300 base pairs in length. The sample of interest - in our case DNA - is fed into the chip with streams of leading electrolyte (LE) and trailing electrolyte (TE). When an electric field is applied, the species migrate based on their electrophoretic mobilities. Because the ions in the leading electrolyte have a high electrophoretic mobility, they race ahead of the slower sample and trailing

  15. UV light induced DNA damages and the radiation protection effects of Lingzi mushroom extract

    International Nuclear Information System (INIS)

    Vo Thi Thuong Lan; Dinh Ba Tuan; Ta Bich Thuan; Tran Bang Diep; Tran Minh Quynh

    2016-01-01

    UV light has strongly influenced on the growth of E. coli as well as caused DNA damages. Configurations of both genomic DNA and pUC 19 plasmids extracted from E. coli were significantly changed by the exposure to UV light of 254 nm and DLT, an extract of Ganoderma lucidum Lingzi mushroom. The results also revealed the radio-protective effects of DLT to UV radiation. By adding 2% DLT to its culturing suspension, the growth of E. coli was significantly decreased, whereas a low DLT amount of about 0.5% slightly improved its growth, indicated that the DLT extract can be used as a promising protective substance against UV radiation. At the molecular level, the radio-protective effects of DLT were observed for both UV treated DNA and protein. Thus, DLT can protect DNA in vivo, but not in vitro. This effect was also observed for Taq polymerase, suggested that the radioprotection effect of DLT may due to it accelerated the degradation of radicals or species that produced in the suspensions during UV exposure. (author)

  16. Assessment of chromium biostabilization in contaminated soils using standard leaching and sequential extraction techniques

    International Nuclear Information System (INIS)

    Papassiopi, Nymphodora; Kontoyianni, Athina; Vaxevanidou, Katerina; Xenidis, Anthimos

    2009-01-01

    The iron reducing microorganism Desulfuromonas palmitatis was evaluated as potential biostabilization agent for the remediation of chromate contaminated soils. D. palmitatis were used for the treatment of soil samples artificially contaminated with Cr(VI) at two levels, i.e. 200 and 500 mg kg -1 . The efficiency of the treatment was evaluated by applying several standard extraction techniques on the soil samples before and after treatment, such as the EN12457 standard leaching test, the US EPA 3060A alkaline digestion method and the BCR sequential extraction procedure. The water soluble chromium as evaluated with the EN leaching test, was found to decrease after the biostabilization treatment from 13 to less than 0.5 mg kg -1 and from 120 to 5.6 mg kg -1 for the soil samples contaminated with 200 and 500 mg Cr(VI) per kg soil respectively. The BCR sequential extraction scheme, although not providing accurate estimates about the initial chromium speciation in contaminated soils, proved to be a useful tool for monitoring the relative changes in element partitioning, as a consequence of the stabilization treatment. After bioreduction, the percentage of chromium retained in the two least soluble BCR fractions, i.e. the 'oxidizable' and 'residual' fractions, increased from 54 and 73% to more than 96% in both soils

  17. Remediation of cadmium-contaminated soil by extraction with para-sulphonato-thiacalix[4]arene, a novel supramolecular receptor

    International Nuclear Information System (INIS)

    Li Yushuang; Hu Xiaojun; Song Xueying; Sun Tieheng

    2012-01-01

    Batch extractions were conducted to evaluate the performance of para-sulphonato-thiacalix[4]arene (STC[4]A), a novel supramolecular receptor, for removing cadmium (Cd) from soil. The extraction mechanism was investigated by determination of the conditional stability constants (log K) of the STC[4]A-Cd complex. The influences of various variables were examined, including pH, contact time, and extractant concentration. The Cd extraction efficiency increased with increasing pH, reaching the maximum at pH 11 and then declining at higher pH values. This pH dependence was explained by the variation in the log K value of the STC[4]A-Cd complex along with pH change. When the STC[4]A dose was increased to an STC[4]A:Cd molar ratio of 2.5:1, Cd was exhaustively removed (up to 96.8%). The comparison experiment revealed that the Cd extraction performance of STC[4]A was almost equivalent to that of EDTA and significantly better than that of natural organic acids. STC[4]A extraction could efficiently prevent co-dissolution of soil minerals. - Highlights: ► First report on para-sulphonato-thiacalix[4]arene (STC[4]A) as extractant for soil washing. ► The Cd extraction performance of STC[4]A was almost equivalent to that of EDTA. ► STC[4]A extraction could efficiently avoid the dissolution of soil minerals, such as K, Ca, Mn. ► Extraction mechanism was investigated by determination of log K values of STC[4]A-Cd complex. ► A rational explanation for the pH dependence of extraction performance was given. - This is the first report on para-sulphonato-thiacalix[4]arene as an extractant for soil washing, which proved to be very efficient for Cd removal and could prevent co-dissolution of soil minerals.

  18. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques.

    OpenAIRE

    Aljanabi, S M; Martinez, I

    1997-01-01

    A very simple, fast, universally applicable and reproducible method to extract high quality megabase genomic DNA from different organisms is described. We applied the same method to extract high quality complex genomic DNA from different tissues (wheat, barley, potato, beans, pear and almond leaves as well as fungi, insects and shrimps' fresh tissue) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even i...

  19. Recovery of environmental analytes from clays and soils by supercritical fluid extracting/gas chromatography

    International Nuclear Information System (INIS)

    Emery, A.P.; Chesler, S.N.; MacCrehan, W.A.

    1992-01-01

    This paper reports on Supercritical Fluid Extraction (SFE) which promises to provide rapid extractions of organic analytes from environmental sample types without the use of hazardous solvents. In addition, SFE protocols using commercial instrumentation can be automated lowering analysis costs. Because of these benefits, we are investigating SFE as an alternative to the solvent extraction (eg. Soxhlet and sonication) techniques required in many EPA test procedures. SFE, using non-polar carbon dioxide as well as more polar supercritical fluids, was used to determine n-alkane hydrocarbons and polynuclear aromatic hydrocarbons (PAHs) in solid samples. The extraction behavior of these analyte classes from environmentally-contaminated soil matrices and model soil and clay matrices was investigated using a SFE apparatus in which the extracted analytes were collected on a solid phase trap and then selectively eluted with a solvent. The SFE conditions for quantitative recovery of n-alkane hydrocarbons in diesel fuel from a series of clays and soils were determined using materials prepared at the 0.02% level with diesel fuel oil in order to simplify analyte collection and analysis after extraction. The effect of extraction parameters including temperature, fluid flow rate and modifier addition were investigated by monitoring the amount of diesel fuel extracted as a function of time

  20. Efficient method for the extraction of genomic DNA from wormwood ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-09-17

    Sep 17, 2008 ... DNA is extracted from plant material it will also contain. *Corresponding author. ... an important issue in the field of plant molecular biology. Various plants ..... An excellent guide to over 500 of the more well known medicinal ...

  1. A rapid and simple method for DNA extraction from yeasts and fungi isolated from Agave fourcroydes.

    Science.gov (United States)

    Tapia-Tussell, Raul; Lappe, Patricia; Ulloa, Miguel; Quijano-Ramayo, Andrés; Cáceres-Farfán, Mirbella; Larqué-Saavedra, Alfonso; Perez-Brito, Daisy

    2006-05-01

    A simple and easy protocol for extracting high-quality DNA from different yeast and filamentous fungal species is described. This method involves two important steps: first, the disruption of cell walls by mechanical means and freezing; and second, the extraction, isolation, and precipitation of genomic DNA. The absorbance ratios (A(260)/A(280)) obtained ranged from 1.6 to 2.0. The main objective of this procedure is to extract pure DNA from yeast and filamentous fungi, including those with high contents of proteins, polysaccharides, and other complex compounds in their cell walls. The yield and quality of the DNAs obtained were suitable for micro/minisatellite primer-polymerase chain reaction (MSP-PCR) fingerprinting as well as for the sequence of the D1/D2 domain of the 26S rDNA.

  2. Characterization of Growing Soil Bacterial Communities across a pH gradient Using H218O DNA-Stable Isotope Probing

    Science.gov (United States)

    Welty-Bernard, A. T.; Schwartz, E.

    2014-12-01

    Recent studies have established consistent relationships between pH and bacterial diversity and community structure in soils from site-specific to landscape scales. However, these studies rely on DNA or PLFA extraction techniques from bulk soils that encompass metabolically active and inactive, or dormant, communities, and loose DNA. Dormant cells may comprise up to 80% of total live cells. If dormant cells dominate a particular environment, it is possible that previous interpretations of the soil variables assumed to drive communities could be profoundly affected. We used H218O stable isotope probing and bar-coded illumina sequencing of 16S rRNA genes to monitor the response of actively growing communities to changes in soil pH in a soil microcosm over 14 days. This substrate-independent approach has several advantages over 13C or 15N-labelled molecules in that all growing bacteria should be able to make use of water, allowing characterization of whole communities. We hypothesized that Acidobacteria would increasingly dominate the growing community and that Actinobacteria and Bacteroidetes would decline, given previously established responses by these taxa to soil pH. Instead, we observed the reverse. Actinobacteria abundance increased three-fold from 26 to 76% of the overall community as soil pH fell from pH 5.6 to pH 4.6. Shifts in community structure and decreases in diversity with declining soil pH were essentially driven by two families, Streptomyceaca and Microbacteracea, which collectively increased from 2 to 40% of the entire community. In contrast, Acidobacteria as a whole declined although numbers of subdivision 1 remained stable across all soil pH levels. We suggest that the brief incubation period in this SIP study selected for growth of acid-tolerant Actinobacteria over Acidobacteria. Taxa within Actinomycetales have been readily cultured over short time frames, suggesting rapid growth patterns. Conversely, taxa within Acidobacteria have been

  3. Extractability of plutonium-238 and curium-242 from a contaminated soil as a function of pH and certain soil components. CH3COOH-NH4OH system

    International Nuclear Information System (INIS)

    Nishita, H.

    1978-01-01

    Extractability of 238 Pu and 242 Cm from an artificially contaminated soil as a function of pH and certain soil components was examined with an equilibrium batch technique by the use of a CH 3 COOH--NH 4 OH extracting system. The influence of various soil components on 238 Pu and 242 Cm extractability was determined indirectly by selective removal of the components from the soil. The soil organic matter appeared to have a major influence on the extractability of these radionuclides. Though to a lesser extent, free iron oxides exerted an influence also. Before removal of soil organic matter, the extractability curves for these radionuclides were qualitatively similar in general form. The nature of this form is discussed. Within the contaminated, untreated soil, the 238 Pu and 242 Cm extractability ranged from 0.60 to 30.8% and 0.11 to 14.83% of dose, respectively, depending on the pH of the extracting solution. The liquid-to soild-phase ratio (K'/sub d/) values ranged from 3.5 x 10 -4 to 2.7 x 10 -2 for 238 Pu and 0.9 x 10 -4 to 1.4 x 10 -2 for 242 Cm. Very low extractability occurred in the pH range from approx. 8.6 to approx. 9.7 for 238 Pu and from 7.6 to approx. 9.7 for 242 Cm

  4. High-pressure extraction of polychlorinated biphenyls from soils and other fine-grained solids

    International Nuclear Information System (INIS)

    Markowz, G.

    1996-12-01

    Four doped and three really contaminated samples were subjected to high-pressure PCB (polychlorinated biphenyl) extraction in a laboratory-scale experimental plant using CO 2 (carbon dioxide) as solvent. The PCB levels (sum out of the six key substances) of the real samples were 2.6, 6.8, and 139 mg/kg. The success of the cleaning process was determined by measuring the residual PCB levels in the soil after the extraction. Parameters were varied and samples were taken selectively from various points in the bed (length 270 mm, diameter 14 mm, weighed - in soil 50-60 g) in order to gain an idea of the effects of upscaling. The following parameters were varied: extraction temperature 40-90 C; extraction pressure 200-300 bar; CO 2 flow rate 3.6-14.6 g/min; CO 2 quantity 0-328 g; degree of contamination (doped samples) 12-60 mg/kg; soil moisture 0-15%; particle size 0-2000 μm; entraining agent methanol, ethanol, acetone; proportion of entraining agent 0-7.5% by weight. Furthermore the influence of moisture at the time of doping on extraction was examined. (orig./ABI) [de

  5. Mobility of radioactive cesium in soil originated from the Fukushima Daiichi nuclear disaster. Application of extraction experiments

    International Nuclear Information System (INIS)

    Yoshikazu Kikawada; Takao Oi; Katsumi Hirose; Masaaki Hirose; Atsushi Tsukamoto; Ko Nakamachi; Teruyuki Honda; Hiroaki Takahashi

    2015-01-01

    Extraction experiments on soil radioactively contaminated by the Fukushima Daiichi Nuclear Power Plant accident were conducted by using a variety of extractants to acquire knowledge on the mobility of radioactive cesium in soil. The experimental results revealed that cesium is tightly bound with soil particles and that radioactive cesium newly deposited on soil due to the accident had apparently a higher mobility than stable cesium commonly existing in soil. The results suggested that radioactive cesium deposited on soil hardly migrates via aqueous processes, although chemical and mineralogical conditions of soil affect their mobility. (author)

  6. Soil acidification increases metal extractability and bioavailability in old orchard soils of Northeast Jiaodong Peninsula in China

    International Nuclear Information System (INIS)

    Li, Lianzhen; Wu, Huifeng; Gestel, Cornelis A.M. van; Peijnenburg, Willie J.G.M.; Allen, Herbert E.

    2014-01-01

    The bioavailability of Cu, Zn, Pb and Cd from field-aged orchard soils in a certified fruit plantation area of the Northeast Jiaodong Peninsula in China was assessed using bioassays with earthworms (Eisenia fetida) and chemical assays. Soil acidity increased with increasing fruit cultivation periods with a lowest pH of 4.34. Metals were enriched in topsoils after decades of horticultural cultivation, with highest concentrations of Cu (132 kg −1 ) and Zn (168 mg kg −1 ) in old apple orchards and Pb (73 mg kg −1 ) and Cd (0.57 mg kg −1 ) in vineyard soil. Earthworm tissue concentrations of Cu and Pb significantly correlated with 0.01 M CaCl 2 -extractable soil concentrations (R 2  = 0.70, p < 0.001 for Cu; R 2  = 0.58, p < 0.01 for Pb). Because of the increased bioavailability, regular monitoring of soil conditions in old orchards and vineyards is recommended, and soil metal guidelines need reevaluation to afford appropriate environmental protection under acidifying conditions. - Highlights: • Soil acidity of Chinese orchards increased with increasing fruit cultivation period. • Metal levels were enriched in topsoils after decades of horticultural cultivation. • Earthworm bioassays and chemical assays were used to assess metal bioavailability. • Earthworm Cu and Pb concentrations correlated with CaCl 2 -extractable concentrations. • Regular monitoring of soil conditions in old orchards and vineyards is recommended. - Long-term cultivation leads to increased acidification and metal accumulation in horticultural soils, with higher metal bioavailability to earthworms

  7. Redox speciation analysis of antimony in soil extracts by hydride generation atomic fluorescence spectrometry

    International Nuclear Information System (INIS)

    Fuentes, Edwar; Pinochet, Hugo; Gregori, Ida de; Potin-Gautier, Martine

    2003-01-01

    A sensitive atomic spectrometric method for the redox speciation analysis of antimony in soils is described. The method is based on the selective generation of stibine from Sb(III) in a continuous flow system using atomic fluorescence spectrometry for detection. Sb(V) is masked by citric or oxalic acid in HCl medium. The procedure was optimized with synthetic solutions of Sb(III) and Sb(V). The effect of carboxylic acid and HCl concentration on the recovery of Sb(III) and Sb(V) species from standard solutions, and on the fluorescence signal were studied. Both species were extracted from soil with H 2 O, 0.05 mol l -1 EDTA and 0.25 mol l -1 H 2 SO 4 . Since the soil samples were collected from sites impacted by copper mining activities, the effect of Cu 2+ on the determination of antimony in synthetic solutions and soil extracts was studied. Cu 2+ decreased the Sb(III) signal, but had no effect on the total antimony determination. Therefore, the selective determination of Sb(III) was carried out in citric acid-HCl medium, using the analyte addition technique. Total antimony in soil extracts was determined using the standard calibration technique after reducing Sb(V) to Sb(III) at room temperature with KI-ascorbic acid. The Sb(V) concentration was calculated from the difference between total antimony and Sb(III). The limits of detection (PS Analytical, Excalibur Millennium model) were 17 and 10 ng l -1 for Sb(III) and total antimony, respectively, and the R.S.D. at the 0.5-μg l -1 level were 2.5 and 2.4%, respectively. The total antimony concentration of soils is in the mg kg -1 range; the Sb recovery from the different soils by the extracting solutions was between less than 0.02% and approximately 10%. Similar recoveries were obtained using EDTA and sulfuric acid solutions. Sb(V) was found to be the main antimony species extracted from soils

  8. The currently used commercial DNA-extraction methods give different results of clostridial and actinobacterial populations derived from human fecal samples.

    Science.gov (United States)

    Maukonen, Johanna; Simões, Catarina; Saarela, Maria

    2012-03-01

    Recently several human health-related microbiota studies have had partly contradictory results. As some differences may be explained by methodologies applied, we evaluated how different storage conditions and commonly used DNA-extraction kits affect bacterial composition, diversity, and numbers of human fecal microbiota. According to our results, the DNA-extraction did not affect the diversity, composition, or quantity of Bacteroides spp., whereas after a week's storage at -20 °C, the numbers of Bacteroides spp. were 1.6-2.5 log units lower (P Eubacterium rectale (Erec)-group, Clostridium leptum group, bifidobacteria, and Atopobium group were 0.5-4 log units higher (P < 0.05) after mechanical DNA-extraction as detected with qPCR, regardless of storage. Furthermore, the bacterial composition of Erec-group differed significantly after different DNA-extractions; after enzymatic DNA-extraction, the most prevalent genera detected were Roseburia (39% of clones) and Coprococcus (10%), whereas after mechanical DNA-extraction, the most prevalent genera were Blautia (30%), Coprococcus (13%), and Dorea (10%). According to our results, rigorous mechanical lysis enables detection of higher bacterial numbers and diversity from human fecal samples. As it was shown that the results of clostridial and actinobacterial populations are highly dependent on the DNA-extraction methods applied, the use of different DNA-extraction protocols may explain the contradictory results previously obtained. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  9. Efficient method for extracting DNA of parasites causing bovine babesiosis from tick vectors

    Science.gov (United States)

    The southern cattle tick, Rhipicephalus (Boophilus) microplus, is an economically important pest costing animal agriculture billions of dollars worldwide. This research focuses on a comparison of three different tick DNA extraction methods: phenol-chloroform extraction (method 1), a modified version...

  10. Comparison of Six DNA Extraction Procedures and the Application of Plastid DNA Enrichment Methods in Selected Non-photosynthetic Plants

    Directory of Open Access Journals (Sweden)

    Shin-Yi Shyu

    2013-12-01

    Full Text Available Genomic DNA was isolated using three DNA extraction commercial kits and three CTAB-based methods for two non-photosynthetic plants, Balanophora japonica and Mitrastemon kanehirai. The quality of the isolated DNA was evaluated and subjected to following restriction enzyme digestions. All six procedures yielded DNA of sufficient quality for PCR, and the method described by Barnwell et al. (1998 performed well in isolating DNA from both species for restriction enzyme digestion. In addition, we succeeded to enrich plastid DNA content by using the methods depending on a high salt buffer to deplete nuclear material. The ‘high salt’ methods based on protocol presented by Milligan (1989 were able to increase plastid DNA effectively and significantly reduce nuclear DNA from M. kanehirai. The plastid DNA enrichment protocols are inexpensive and not time-consuming, and may be applicable to other non-photosynthetic plants.

  11. Use of aqueous and solvent extraction to assess risk and bioavailability of contaminated soil

    International Nuclear Information System (INIS)

    Bordelon, N.; Huebner, H.; Washburn, K.; Donnelly, K.C.

    1995-01-01

    Contaminated media at Superfund sites typically consist of complex mixtures of organic and inorganic chemicals. These mixtures are difficult to characterize, both analytically and toxicologically, especially the complex mixtures of polycyclic aromatic hydrocarbons. The current approach to risk assessment assumes that all contaminants in the soil are available for human exposure. EPA protocol uses solvent extraction to remove chemicals from the soil as a basis for estimating risk to the human population. However, contaminants that can be recovered with a solvent extract may not represent chemicals that are available for exposure. A system using aqueous extraction provides a more realistic picture of what chemicals are bioavailable through leaching and ingestion. A study was conducted with coal tar contaminated soil spiked with benzo(a)pyrene, and trinitrotoluene. Samples were extracted with hexane:acetone and water titrated to pH 2 and pH 7. HPLC analysis demonstrated up to 35% and 29% recovery of contaminants from aqueous extracts with an estimated cancer risk one order of magnitude less than that for solvent extracts. Analysis using the Salmonella/microsome assay showed that solvent extracts were genotoxic with metabolic activation while aqueous extracts showed no genotoxicity. These results suggest that aqueous extraction may be useful in determining what contaminants are available for human exposure, as well as what compounds may pose a risk to human health

  12. Enhanced Attenuation Technologies: Passive Soil Vapor Extraction

    Energy Technology Data Exchange (ETDEWEB)

    Vangelas, K.; Looney, B.; Kamath, R.; Adamson, D.; Newell, C.

    2010-03-15

    Passive soil vapor extraction (PSVE) is an enhanced attenuation (EA) approach that removes volatile contaminants from soil. The extraction is driven by natural pressure gradients between the subsurface and atmosphere (Barometric Pumping), or by renewable sources of energy such as wind or solar power (Assisted PSVE). The technology is applicable for remediating sites with low levels of contamination and for transitioning sites from active source technologies such as active soil vapor extraction (ASVE) to natural attenuation. PSVE systems are simple to design and operate and are more cost effective than active systems in many scenarios. Thus, PSVE is often appropriate as an interim-remedial or polishing strategy. Over the past decade, PSVE has been demonstrated in the U.S. and in Europe. These demonstrations provide practical information to assist in selecting, designing and implementing the technology. These demonstrations indicate that the technology can be effective in achieving remedial objectives in a timely fashion. The keys to success include: (1) Application at sites where the residual source quantities, and associated fluxes to groundwater, are relatively low; (2) Selection of the appropriate passive energy source - barometric pumping in cases with a deep vadose zone and barrier (e.g., clay) layers that separate the subsurface from the atmosphere and renewable energy assisted PSVE in other settings and where higher flow rates are required. (3) Provision of sufficient access to the contaminated vadose zones through the spacing and number of extraction wells. This PSVE technology report provides a summary of the relevant technical background, real-world case study performance, key design and cost considerations, and a scenario-based cost evaluation. The key design and cost considerations are organized into a flowchart that dovetails with the Enhanced Attenuation: Chlorinated Organics Guidance of the Interstate Technology and Regulatory Council (ITRC). The PSVE

  13. DNA extraction from coral reef sediment bacteria for the polymerase chain reaction.

    Science.gov (United States)

    Guthrie, J N; Moriarty, D J; Blackall, L L

    2000-12-15

    A rapid and effective method for the direct extraction of high molecular weight amplifiable DNA from two coral reef sediments was developed. DNA was amplified by the polymerase chain reaction (PCR) using 16S rDNA specific primers. The amplicons were digested with HaeIII, HinP1I and MspI and separated using polyacrylamide gel electrophoresis and silver staining. The resulting amplified ribosomal DNA restriction analysis (ARDRA) patterns were used as a fingerprint to discern differences between the coral reef sediment samples. Results indicated that ARDRA is an effective method for determining differences within the bacterial community amongst different environmental samples.

  14. MICHIGAN SOIL VAPOR EXTRACTION REMEDIATION (MISER) MODEL: A COMPUTER PROGRAM TO MODEL SOIL VAPORT EXTRACTION AND BIOVENTING OF ORGANIC MATERIALS IN UNSATURATED GEOLOGICAL MATERIAL

    Science.gov (United States)

    This report describes the formulation, numerical development, and use of a multiphase, multicomponent, biodegradation model designed to simulate physical, chemical, and biological interactions occurring primarily in field scale soil vapor extraction (SVE) and bioventing (B...

  15. Root water extraction and limiting soil hydraulic conditions estimated by numerical simulation

    NARCIS (Netherlands)

    Jong van Lier, de Q.; Metselaar, K.; Dam, van J.C.

    2006-01-01

    Root density, soil hydraulic functions, and hydraulic head gradients play an important role in the determination of transpiration-rate-limiting soil water contents. We developed an implicit numerical root water extraction model to solve the Richards equation for the modeling of radial root water

  16. Selective dissolution followed by EDDS washing of an e-waste contaminated soil: Extraction efficiency, fate of residual metals, and impact on soil environment.

    Science.gov (United States)

    Beiyuan, Jingzi; Tsang, Daniel C W; Valix, Marjorie; Zhang, Weihua; Yang, Xin; Ok, Yong Sik; Li, Xiang-Dong

    2017-01-01

    To enhance extraction of strongly bound metals from oxide minerals and organic matter, this study examined the sequential use of reductants, oxidants, alkaline solvents and organic acids followed by a biodegradable chelating agent (EDDS, [S,S]-ethylene-diamine-disuccinic-acid) in a two-stage soil washing. The soil was contaminated by Cu, Zn, and Pb at an e-waste recycling site in Qingyuan city, China. In addition to extraction efficiency, this study also examined the fate of residual metals (e.g., leachability, bioaccessibility, and distribution) and the soil quality parameters (i.e., cytotoxicity, enzyme activities, and available nutrients). The reductants (dithionite-citrate-bicarbonate and hydroxylamine hydrochloride) effectively extracted metals by mineral dissolution, but elevated the leachability and bioaccessibility of metals due to the transformation from Fe/Mn oxides to labile fractions. Subsequent EDDS washing was found necessary to mitigate the residual risks. In comparison, prior washing by oxidants (persulphate, hypochlorite, and hydrogen peroxide) was marginally useful because of limited amount of soil organic matter. Prior washing by alkaline solvents (sodium hydroxide and sodium bicarbonate) was also ineffective due to metal precipitation. In contrast, prior washing by low-molecular-weight organic acids (citrate and oxalate) improved the extraction efficiency. Compared to hydroxylamine hydrochloride, citrate and oxalate induced lower cytotoxicity (Microtox) and allowed higher enzyme activities (dehydrogenase, acid phosphatase, and urease) and soil nutrients (available nitrogen and phosphorus), which would facilitate reuse of the treated soil. Therefore, while sequential washing proved to enhance extraction efficacy, the selection of chemical agents besides EDDS should also include the consideration of effects on metal leachability/bioaccessibility and soil quality. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Evaluating the efficacy of a centrifugation-flotation method for extracting Ascaris ova from soil

    DEFF Research Database (Denmark)

    Cranston, Imogen; Teoh, Penelope J.; baker, Sarah M.

    2016-01-01

    method to extract STH ova from soil makes it challenging to examine whether the use of latrines may or may not have an effect on environmental contamination with ova. The present study evaluated the recovery rate of a method developed to extract STH ova from soil. Methods: The adapted centrifugation...... with increasing soil moisture content, particle size and organic matter content. The association between recovery rate and organic matter content was statistically significant. Conclusions: The present study identified a low recovery rate for an adapted centrifugation-flotation method, although this was similar...

  18. Estimating the impact of seawater on the production of soil water-extractable organic carbon during coastal erosion.

    Science.gov (United States)

    Dou, Fugen; Ping, Chien-Lu; Guo, Laodong; Jorgenson, Torre

    2008-01-01

    The production of water-extractable organic carbon (WEOC) during arctic coastal erosion and permafrost degradation may contribute significantly to C fluxes under warming conditions, but it remains difficult to quantify. A tundra soil collected near Barrow, AK, was selected to evaluate the effects of soil pretreatments (oven drying vs. freeze drying) as well as extraction solutions (pure water vs. seawater) on WEOC yields. Both oven drying and freeze drying significantly increased WEOC release compared with the original moist soil samples; dried samples released, on average, 18% more WEOC than did original moist samples. Similar results were observed for the production of low-molecular-weight dissolved organic C. However, extractable OC released from different soil horizons exhibited differences in specific UV absorption, suggesting differences in WEOC quality. Furthermore, extractable OC yields were significantly less in samples extracted with seawater compared with those extracted with pure water, likely due to the effects of major ions on extractable OC flocculation. Compared with samples from the active horizons, upper permafrost samples released more WEOC, suggesting that continuously frozen samples were more sensitive than samples that had experienced more drying-wetting cycles in nature. Specific UV absorption of seawater-extracted OC was significantly lower than that of OC extracted using pure water, suggesting more aromatic or humic substances were flocculated during seawater extraction. Our results suggest that overestimation of total terrestrial WEOC input to the Arctic Ocean during coastal erosion could occur if estimations were based on WEOC extracted from dried soil samples using pure water.

  19. Extractability of 137Cs in Response to its Input Forms into Fukushima Forest Soils.

    Science.gov (United States)

    Mengistu, T. T.; Carasco, L.; Orjollet, D.; Coppin, F.

    2017-12-01

    In case of nuclear accidents like Fukushima disaster, the influence of 137Cs depositional forms (soluble and/or solid forms) on mineral soil of forest environment on its availability have not reported yet. Soluble (137Cs tagged ultra-pure water) and solid (137Cs contaminated litter-OL and fragmented litter-OF) input forms were mixed with the mineral soils collected under Fukushima coniferous and broadleaf forests. The mixtures then incubated under controlled laboratory condition to evaluate the extractability of 137Cs in soil over time in the presence of decomposition process through two extracting reagents- water and ammonium acetate. Results show that extracted 137Cs fraction with water was less than 1% for soluble input form and below detection limit for solid input form. On the same way with acetate reagent, the extracted 137Cs fraction ranged from 46 to 56% for soluble input and 2 to 15% for solid input, implying the nature of 137Cs contamination strongly influences the extractability and hence the mobility of 137Cs in soil. Although the degradation rate of the organic materials has been calculated in the range of 0.18 ± 0.1 to 0.24 ± 0.1 y-1, its impact on 137Cs extractability appeared very weak at least within the observation period, probably due to shorter time scale. Concerning the treatments of solid 137Cs input forms through acetate extraction, relatively more 137Cs has been extracted from broadleaf organic materials mixes (BL-OL & BL-OF) than the coniferous counterparts. This probably is due to the fact that the lignified coniferous organic materials (CED-OL & CED-OF) components tend to retain more 137Cs than that of the broadleaf. Generally, by extrapolating these observations in to a field context, one can expect more available 137Cs fraction in forest soil from wet depositional pathways such as throughfall and stemflow than those attached with organic materials like litter (OL) and its eco-processed forms (OF).

  20. Antifungal activity of root, bark, leaf and soil extracts of Androstachys ...

    African Journals Online (AJOL)

    Extracts of leaf, root, soil and bark of Androstachys johnsonii Prain (commonly called Lembobo ironwood) screened for antifungal activity had a significant inhibitory effect on the most of fungi tested in this investigation. Of the four fungi tested in the present study Fusarium solani was significantly inhibited by all extracts (that ...

  1. Soil pretreatment and fast cell lysis for direct polymerase chain reaction from forest soils for terminal restriction fragment length polymorphism analysis of fungal communities

    Directory of Open Access Journals (Sweden)

    Fei Cheng

    Full Text Available Abstract Humic substances in soil DNA samples can influence the assessment of microbial diversity and community composition. Using multiple steps during or after cell lysis adds expenses, is time-consuming, and causes DNA loss. A pretreatment of soil samples and a single step DNA extraction may improve experimental results. In order to optimize a protocol for obtaining high purity DNA from soil microbiota, five prewashing agents were compared in terms of their efficiency and effectiveness in removing soil contaminants. Residual contaminants were precipitated by adding 0.6 mL of 0.5 M CaCl2. Four cell lysis methods were applied to test their compatibility with the pretreatment (prewashing + Ca2+ flocculation and to ultimately identify the optimal cell lysis method for analyzing fungal communities in forest soils. The results showed that pretreatment with TNP + Triton X-100 + skim milk (100 mM Tris, 100 mM Na4P2O7, 1% polyvinylpyrrolidone, 100 mM NaCl, 0.05% Triton X-100, 4% skim milk, pH 10.0 removed most soil humic contaminants. When the pretreatment was combined with Ca2+ flocculation, the purity of all soil DNA samples was further improved. DNA samples obtained by the fast glass bead-beating method (MethodFGB had the highest purity. The resulting DNA was successfully used, without further purification steps, as a template for polymerase chain reaction targeting fungal internal transcribed spacer regions. The results obtained by terminal restriction fragment length polymorphism analysis indicated that the MethodFGB revealed greater fungal diversity and more distinctive community structure compared with the other methods tested. Our study provides a protocol for fungal cell lysis in soil, which is fast, convenient, and effective for analyzing fungal communities in forest soils.

  2. Influence of the soil/solution ratio, interaction time, and extractant on the evaluation of iron chelate sorption/desorption by soils.

    Science.gov (United States)

    Hernández-Apaolaza, Lourdes; Lucena, Juan J

    2011-03-23

    Synthetic Fe chelates are the most efficient agricultural practice to control Fe deficiency in crops, EDTA/Fe3+ and o,o-EDDHA/Fe3+ being the most commonly used. Their efficacy as Fe sources and carriers in soils can be severely limited by their retention on it. The aim of this work is to evaluate the possible bias introduced in the studies of the iron chelate retention by soils. For that purpose, results obtained for EDTA and EDDHA iron chelates from two batch studies with different soil/solution ratios were compared with data obtained for a leaching column experiment. Moreover, different extractants were tested to study the o,o-EDDHA/Fe3+ and o,p-EDDHA/Fe3+ desorption from a calcareous soil, and also the effect of the interaction time in their retention process has been evaluated. In summary, the mobility through a calcareous soil of the studied iron chelates differs greatly depending on the type of iron chelate and also on the procedure used to evaluate the retention and the soil/solution ratio used. In general, the leaching column method is preferred because the achieved conclusions are more representative of the natural conditions, but batch methods are very useful as a preliminary experiment, especially one with a high soil/solution ratio. The iron chelate desorption could be quantified by using a sequential extraction with water, sodium sulfate, and DTPA as extractants. Under the experimental conditions used in this study, o,o-EDDHA/Fe3+ retention increased with interaction time.

  3. High-throughput diagnosis of potato cyst nematodes in soil samples.

    Science.gov (United States)

    Reid, Alex; Evans, Fiona; Mulholland, Vincent; Cole, Yvonne; Pickup, Jon

    2015-01-01

    Potato cyst nematode (PCN) is a damaging soilborne pest of potatoes which can cause major crop losses. In 2010, a new European Union directive (2007/33/EC) on the control of PCN came into force. Under the new directive, seed potatoes can only be planted on land which has been found to be free from PCN infestation following an official soil test. A major consequence of the new directive was the introduction of a new harmonized soil sampling rate resulting in a threefold increase in the number of samples requiring testing. To manage this increase with the same staffing resources, we have replaced the traditional diagnostic methods. A system has been developed for the processing of soil samples, extraction of DNA from float material, and detection of PCN by high-throughput real-time PCR. Approximately 17,000 samples are analyzed each year using this method. This chapter describes the high-throughput processes for the production of float material from soil samples, DNA extraction from the entire float, and subsequent detection and identification of PCN within these samples.

  4. Use of real-time PCR to evaluate two DNA extraction methods from food

    Directory of Open Access Journals (Sweden)

    Maria Regina Branquinho

    2012-03-01

    Full Text Available The DNA extraction is a critical step in Genetically Modified Organisms analysis based on real-time PCR. In this study, the CTAB and DNeasy methods provided good quality and quantity of DNA from the texturized soy protein, infant formula, and soy milk samples. Concerning the Certified Reference Material consisting of 5% Roundup Ready® soybean, neither method yielded DNA of good quality. However, the dilution test applied in the CTAB extracts showed no interference of inhibitory substances. The PCR efficiencies of lectin target amplification were not statistically different, and the coefficients of correlation (R² demonstrated high degree of correlation between the copy numbers and the threshold cycle (Ct values. ANOVA showed suitable adjustment of the regression and absence of significant linear deviations. The efficiencies of the p35S amplification were not statistically different, and all R² values using DNeasy extracts were above 0.98 with no significant linear deviations. Two out of three R² values using CTAB extracts were lower than 0.98, corresponding to lower degree of correlation, and the lack-of-fit test showed significant linear deviation in one run. The comparative analysis of the Ct values for the p35S and lectin targets demonstrated no statistical significant differences between the analytical curves of each target.

  5. Characterization of soil nematode communities in three cropping systems through morphological and DNA metabarcoding approaches

    Science.gov (United States)

    Communities of soil nematodes impact ecosystem functions, including plant growth, decomposition, and nutrient cycling, all of which are vital processes in agriculture. We used complementary morphological and DNA metabarcoding analyses to characterize soil nematode communities in three cropping syste...

  6. Effects of lead mineralogy on soil washing enhanced by ferric salts as extracting and oxidizing agents.

    Science.gov (United States)

    Yoo, Jong-Chan; Park, Sang-Min; Yoon, Geun-Seok; Tsang, Daniel C W; Baek, Kitae

    2017-10-01

    In this study, we evaluated the feasibility of using ferric salts including FeCl 3 and Fe(NO 3 ) 3 as extracting and oxidizing agents for a soil washing process to remediate Pb-contaminated soils. We treated various Pb minerals including PbO, PbCO 3 , Pb 3 (CO 3 ) 2 (OH) 2 , PbSO 4 , PbS, and Pb 5 (PO 4 ) 3 (OH) using ferric salts, and compared our results with those obtained using common washing agents of HCl, HNO 3 , disodium-ethylenediaminetetra-acetic acid (Na 2 -EDTA), and citric acid. The use of 50 mM Fe(NO 3 ) 3 extracted significantly more Pb (above 96% extraction) from Pb minerals except PbSO 4 (below 55% extraction) compared to the other washing agents. In contrast, washing processes using FeCl 3 and HCl were not effective for extraction from Pb minerals because of PbCl 2 precipitation. Yet, the newly formed PbCl 2 could be dissolved by subsequent wash with distilled water under acidic conditions. When applying our washing method to remediate field-contaminated soil from a shooting range that had high concentrations of Pb 3 (CO 3 ) 2 (OH) 2 and PbCO 3 , we extracted more Pb (approximately 99% extraction) from the soil using 100 mM Fe(NO 3 ) 3 than other washing agents at the same process conditions. Our results show that ferric salts can be alternative washing agents for Pb-contaminated soils in view of their extracting and oxidizing abilities. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. The Effect of Storage and Extraction Methods on Amplification of Plasmodium falciparum DNA from Dried Blood Spots

    NARCIS (Netherlands)

    Schwartz, A.; Baidjoe, A.Y.; Rosenthal, P.J.; Dorsey, G.; Bousema, T.; Greenhouse, B.

    2015-01-01

    Extraction and amplification of DNA from dried blood spots (DBS) collected in field studies is commonly used for detection of Plasmodium falciparum. However, there have been few systematic efforts to determine the effects of storage and extraction methods on the sensitivity of DNA amplification. We

  8. Evaluation of methods to improve the extraction and recovery of DNA from cotton swabs for forensic analysis.

    Science.gov (United States)

    Adamowicz, Michael S; Stasulli, Dominique M; Sobestanovich, Emily M; Bille, Todd W

    2014-01-01

    Samples for forensic DNA analysis are often collected from a wide variety of objects using cotton or nylon tipped swabs. Testing has shown that significant quantities of DNA are retained on the swab, however, and subsequently lost. When processing evidentiary samples, the recovery of the maximum amount of available DNA is critical, potentially dictating whether a usable profile can be derived from a piece of evidence or not. The QIAamp DNA Investigator extraction kit was used with its recommended protocol for swabs (one hour incubation at 56°C) as a baseline. Results indicate that over 50% of the recoverable DNA may be retained on the cotton swab tip, or otherwise lost, for both blood and buccal cell samples when using this protocol. The protocol's incubation time and temperature were altered, as was incubating while shaking or stationary to test for increases in recovery efficiency. An additional step was then tested that included periodic re-suspension of the swab tip in the extraction buffer during incubation. Aliquots of liquid blood or a buccal cell suspension were deposited and dried on cotton swabs and compared with swab-less controls. The concentration of DNA in each extract was quantified and STR analysis was performed to assess the quality of the extracted DNA. Stationary incubations and those performed at 65°C did not result in significant gains in DNA yield. Samples incubated for 24 hours yielded less DNA. Increased yields were observed with three and 18 hour incubation periods. Increases in DNA yields were also observed using a swab re-suspension method for both cell types. The swab re-suspension method yielded an average two-fold increase in recovered DNA yield with buccal cells and an average three-fold increase with blood cells. These findings demonstrate that more of the DNA collected on swabs can be recovered with specific protocol alterations.

  9. Contribution of microorganisms to non-extractable residue formation during biodegradation of ibuprofen in soil

    Energy Technology Data Exchange (ETDEWEB)

    Nowak, Karolina M., E-mail: karolina.nowak@ufz.de [UFZ, Helmholtz Centre for Environmental Research, Department of Environmental Biotechnology, Permoserstraße 15, 04318 Leipzig (Germany); Department of Environmental Biology and Chemodynamics, Institute for Environmental Research (Biology V), RWTH Aachen University, Worringerweg 1, 52074 Aachen (Germany); Girardi, Cristobal; Miltner, Anja [UFZ, Helmholtz Centre for Environmental Research, Department of Environmental Biotechnology, Permoserstraße 15, 04318 Leipzig (Germany); Gehre, Matthias [UFZ, Helmholtz Centre for Environmental Research, Department of Isotope Biogeochemistry, Permoserstraße 15, 04318 Leipzig (Germany); Schäffer, Andreas [Department of Environmental Biology and Chemodynamics, Institute for Environmental Research (Biology V), RWTH Aachen University, Worringerweg 1, 52074 Aachen (Germany); Kästner, Matthias [UFZ, Helmholtz Centre for Environmental Research, Department of Environmental Biotechnology, Permoserstraße 15, 04318 Leipzig (Germany)

    2013-02-15

    Non-extractable residues (NER) formed during biodegradation of organic contaminants in soil are considered to be mainly composed of parent compounds or their primary metabolites with hazardous potential. However, in the case of biodegradable organic compounds, the soil NER may also contain microbial biomass components, for example fatty acids (FA) and amino acids (AA). After cell death, these biomolecules are subsequently incorporated into non-living soil organic matter (SOM) and are stabilised ultimately forming hardly extractable residues of biogenic origin. We investigated biodegradation of {sup 13}C{sub 6}-ibuprofen, in particular the metabolic incorporation of the {sup 13}C-label into FA and AA and their fate in soil over 90 days. {sup 13}C-FA and {sup 13}C-AA amounts in the living microbial biomass fraction initially increased, then decreased over time and were continuously incorporated into the non-living SOM pool. The {sup 13}C-FA in the non-living SOM remained stable from day 59 whereas the contents of {sup 13}C-AA slightly increased until the end. After 90 days, nearly all NER were biogenic as they were made up almost completely by natural biomass compounds. The presented data demonstrated that the potential environmental risks related to the ibuprofen-derived NER are overestimated. - Highlights: ► Biogenic residue formation during microbial degradation of ibuprofen was studied. ► Nearly all non-extractable residues derived from ibuprofen were biogenic. ► Fatty acids and amino acids formed biogenic non-extractable residues and were stabilised in soil. ► Environmental risks of ibuprofen-derived non-extractable residues are overestimated.

  10. Contribution of microorganisms to non-extractable residue formation during biodegradation of ibuprofen in soil

    International Nuclear Information System (INIS)

    Nowak, Karolina M.; Girardi, Cristobal; Miltner, Anja; Gehre, Matthias; Schäffer, Andreas; Kästner, Matthias

    2013-01-01

    Non-extractable residues (NER) formed during biodegradation of organic contaminants in soil are considered to be mainly composed of parent compounds or their primary metabolites with hazardous potential. However, in the case of biodegradable organic compounds, the soil NER may also contain microbial biomass components, for example fatty acids (FA) and amino acids (AA). After cell death, these biomolecules are subsequently incorporated into non-living soil organic matter (SOM) and are stabilised ultimately forming hardly extractable residues of biogenic origin. We investigated biodegradation of 13 C 6 -ibuprofen, in particular the metabolic incorporation of the 13 C-label into FA and AA and their fate in soil over 90 days. 13 C-FA and 13 C-AA amounts in the living microbial biomass fraction initially increased, then decreased over time and were continuously incorporated into the non-living SOM pool. The 13 C-FA in the non-living SOM remained stable from day 59 whereas the contents of 13 C-AA slightly increased until the end. After 90 days, nearly all NER were biogenic as they were made up almost completely by natural biomass compounds. The presented data demonstrated that the potential environmental risks related to the ibuprofen-derived NER are overestimated. - Highlights: ► Biogenic residue formation during microbial degradation of ibuprofen was studied. ► Nearly all non-extractable residues derived from ibuprofen were biogenic. ► Fatty acids and amino acids formed biogenic non-extractable residues and were stabilised in soil. ► Environmental risks of ibuprofen-derived non-extractable residues are overestimated

  11. Permanganate-assisted removal of PCR inhibitors during the DNA Chelex extraction from stained denim samples.

    Science.gov (United States)

    Pîrlea, Sorina; Puiu, Mihaela; Răducan, Adina; Oancea, Dumitru

    2017-03-01

    In this study, it was demonstrated that the DNA Chelex extraction combined with the permanganate assisted-oxidation is highly efficient in removing the PCR inhibitors often found in clothing materials, such as phthalocyanine. The extraction assays were conducted in saliva, blood and epithelial cells samples mixed with three oxidation-resistant dye copper(II) α-phthalocyanine, copper(II) β-phthalocyanine and tetrasulfonated copper(II) β-phthalocyanine. After DNA amplification, all samples were able to provide full DNA profiles. The permanganate/Chelex system was tested further on denim-stained samples and displayed the same ability to remove the PCR inhibitors from the commercial textile materials.

  12. Effects of some organic materials on bicarbonate extractable phosphate content of soils having different pH

    Directory of Open Access Journals (Sweden)

    Nutullah Özdemir

    2016-10-01

    Full Text Available This study was carried out to determine the effects of rice husk compost (RC, town waste compost (TW and tobacco waste (TB on bicarbonate extractable phosphate content (P in soils having different pH levels under greenhouse conditions. Soil samples used in this study were taken from surfaces (0-20 cm of agricultural fields around Samsun, Northern Anatolia. The experiment was conducted according to split plot design with four doses of organic matterials (0, 2.5, 5.0 and 7.5, %. After a month of mixing organic matterials into soils, lettuce were grown in the medias. According to the results, RC, TW and TB applications into acidic (Tepecik, neutral (Kampüs and alkaline (Çetinkaya soils increased extractable P content. It was observed that effectiveness of organic matterials changed depend on soil reaction, type and dose of organic matterials. All organic wastes were more effective on increment of bicarbonate extractable phosphate content in neutral soil pH when compared the other soil pH levels.

  13. Rapid DNA extraction from dried blood spots on filter paper: potential applications in biobanking.

    Science.gov (United States)

    Choi, Eun-Hye; Lee, Sang Kwang; Ihm, Chunhwa; Sohn, Young-Hak

    2014-12-01

    Dried blood spot (DBS) technology is a microsampling alternative to traditional plasma or serum sampling for pharmaco- or toxicokinetic evaluation. DBS technology has been applied to diagnostic screening in drug discovery, nonclinical, and clinical settings. We have developed an improved elution protocol involving boiling of blood spots dried on Whatman filter paper. The purpose of this study was to compare the quality, purity, and quantity of DNA isolated from frozen blood samples and DBSs. We optimized a method for extraction and estimation of DNA from blood spots dried on filter paper (3-mm FTA card). A single DBS containing 40 μL blood was used. DNA was efficiently extracted in phosphate-buffered saline (PBS) or Tris-EDTA (TE) buffer by incubation at 37°C overnight. DNA was stable in DBSs that were stored at room temperature or frozen. The housekeeping genes GAPDH and beta-actin were used as positive standards for polymerase chain reaction (PCR) validation of general diagnostic screening. Our simple and convenient DBS storage and extraction methods are suitable for diagnostic screening by using very small volumes of blood collected on filter paper, and can be used in biobanks for blood sample storage.

  14. Comparison of six simple methods for extracting ribosomal and mitochondrial DNA from Toxocara and Toxascaris nematodes.

    Science.gov (United States)

    Mikaeili, F; Kia, E B; Sharbatkhori, M; Sharifdini, M; Jalalizand, N; Heidari, Z; Zarei, Z; Stensvold, C R; Mirhendi, H

    2013-06-01

    Six simple methods for extraction of ribosomal and mitochondrial DNA from Toxocara canis, Toxocara cati and Toxascaris leonina were compared by evaluating the presence, appearance and intensity of PCR products visualized on agarose gels and amplified from DNA extracted by each of the methods. For each species, two isolates were obtained from the intestines of their respective hosts: T. canis and T. leonina from dogs, and T. cati from cats. For all isolates, total DNA was extracted using six different methods, including grinding, boiling, crushing, beating, freeze-thawing and the use of a commercial kit. To evaluate the efficacy of each method, the internal transcribed spacer (ITS) region and the cytochrome c oxidase subunit 1 (cox1) gene were chosen as representative markers for ribosomal and mitochondrial DNA, respectively. Among the six DNA extraction methods, the beating method was the most cost effective for all three species, followed by the commercial kit. Both methods produced high intensity bands on agarose gels and were characterized by no or minimal smear formation, depending on gene target; however, beating was less expensive. We therefore recommend the beating method for studies where costs need to be kept at low levels. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. An altered Pseudomonas diversity is recovered from soil by using nutrient-poor Pseudomonas-selective soil extract media

    DEFF Research Database (Denmark)

    Aagot, N.; Nybroe, O.; Nielsen, P.

    2001-01-01

    We designed five Pseudomonas-selective soil extract NAA media containing the selective properties of trimethoprim and sodium lauroyl sarcosine and 0 to 100% of the amount of Casamino Acids used in the classical Pseudomonas-selective Gould's S1 medium. All of the isolates were confirmed to be Pseu......We designed five Pseudomonas-selective soil extract NAA media containing the selective properties of trimethoprim and sodium lauroyl sarcosine and 0 to 100% of the amount of Casamino Acids used in the classical Pseudomonas-selective Gould's S1 medium. All of the isolates were confirmed....... Several of these analyses showed that the amount of Casamino Acids significantly influenced the diversity of the recovered Pseudomonas isolates. Furthermore, the data suggested that specific Pseudomonas subpopulations were represented on the nutrient-poor media. The NAA 1:100 medium, containing ca. 15 mg...... of organic carbon per liter, consistently gave significantly higher Pseudomonas CFU counts than Gould's S1 when tested on four Danish soils. NAA 1:100 may, therefore, be a better medium than Gould's S1 for enumeration and isolation of Pseudomonas from the low-nutrient soil environment....

  16. DNA extraction in Echinococcus granulosus and Taenia spp. eggs in dogs stool samples applying thermal shock.

    Science.gov (United States)

    Hidalgo, Alejandro; Melo, Angélica; Romero, Fernando; Hidalgo, Víctor; Villanueva, José; Fonseca-Salamanca, Flery

    2018-03-01

    The extraction of DNA in taeniid eggs shows complications attached to the composition of stool samples and the high resistance of eggs to degradation. The objective of this study was to test a method of DNA extraction in taeniid eggs by applying a thermal shock to facilitate the chemical-enzymatic degradation of these elements. A group of six tubes containing 1 ml of dog stool sample was spiked with eggs of Echinococcus granulosus and another group of six with Taenia pisiformis. Samples were floated with supersaturated sugar solution and centrifuged. The upper portion of each tube (500 μl) was aspirated and deposited in 1.5 ml tubes. Three tubes from each group were incubated at -20 °C and then at 90 °C, the remaining three from each group, incubated at room temperature. Proteinase K and lysis buffer were added to each tube and incubated for 12 h at 58 °C. The lysis effect was evaluated by microscopy at 3, 6 and 12 h and integrity by electrophoresis in 1% agarose gels. With the same experimental scheme, the thermal shock effect was evaluated in extractions of 1, 2, 3 and 4 eggs of each species and the DNA was quantified. Additionally, the protocol was applied in samples of 4 dogs diagnosed with natural infection by Taeniidae worms. Finally, all the extractions were tested by PCR amplification. Both E. granulosus and T. pisiformis eggs showed a similar response in the tests. In samples without treatment, the lysis effect was poor and showed no differences over time, but in those subjected to thermal shock, eggs degradation increased with time. In both treatments, there was no DNA loss integrity. The protocol applied to limited amounts of eggs yielded PCR products in 100% of the samples exposed to thermal shock, allowing PCR amplifications up to 1 egg. In non-exposed samples, the results were not replicable. However, DNA quantification showed low values in both treatments. In turn, DNA extractions with thermal shock in infected dog samples

  17. Occurrence of non extractable pesticide residues in physical and chemical fractions of two soils

    Science.gov (United States)

    Andreou, Kostas; Semple, Kirk; Jones, Kevin

    2010-05-01

    Soils are considered to be a significant sink for organic contaminants, including pesticides, in the environment. Understanding the distribution and localisation of aged pesticide residues in soil is of great importance for assessing the mobility and availability of these chemicals in the environment. This study aimed to characterise the distribution of radiolabeled herbicide isoproturon and the radiolabeled insecticides diazinon and cypermethrin in two organically managed soils. The soils were spiked and aged under laboratory conditions for 17 months. The labile fraction of the pesticides residues was recovered in CaCl2 (0.01M) and then subjected to physical size fractionation using sedimentation and centrifugation steps, with >20μm, 20-2μm and 2-0.1μm soil factions collected. Further, the distribution of the pesticide residues in the organic matter of the fractionated soil was investigated using a sequential alkaline extraction (0.1N NaOH) into humic and fulvic acid and humin. Soil fractions of 20-2μm and 2-0.1μm had the largest burden of the 14C-residues. Different soil constituents have different capacities to form non-extractable residues. Soil solid fractions of 20-2 µm and 20 µm). Fulvic acid showed to play a vital role in the formation and stabilisation of non-extractable 14C-pesticide residues in most cases.Assessment of the likelihood of the pesticide residues to become available to soil biota requires an understanding of the structure of the SOM matrix and the definition of the kinetics of the pesticide residues in different SOM pools as a function of the time.

  18. Rapid methods for the extraction and archiving of molecular grade fungal genomic DNA.

    Science.gov (United States)

    Borman, Andrew M; Palmer, Michael; Johnson, Elizabeth M

    2013-01-01

    The rapid and inexpensive extraction of fungal genomic DNA that is of sufficient quality for molecular approaches is central to the molecular identification, epidemiological analysis, taxonomy, and strain typing of pathogenic fungi. Although many commercially available and in-house extraction procedures do eliminate the majority of contaminants that commonly inhibit molecular approaches, the inherent difficulties in breaking fungal cell walls lead to protocols that are labor intensive and that routinely take several hours to complete. Here we describe several methods that we have developed in our laboratory that allow the extremely rapid and inexpensive preparation of fungal genomic DNA.

  19. Development of a real-world direct interface for integrated DNA extraction and amplification in a microfluidic device.

    Science.gov (United States)

    Shaw, Kirsty J; Joyce, Domino A; Docker, Peter T; Dyer, Charlotte E; Greenway, Gillian M; Greenman, John; Haswell, Stephen J

    2011-02-07

    Integrated DNA extraction and amplification have been carried out in a microfluidic device using electro-osmotic pumping (EOP) for fluidic control. All the necessary reagents for performing both DNA extraction and polymerase chain reaction (PCR) amplification were pre-loaded into the microfluidic device following encapsulation in agarose gel. Buccal cells were collected using OmniSwabs [Whatman™, UK] and manually added to a chaotropic binding/lysis solution pre-loaded into the microfluidic device. The released DNA was then adsorbed onto a silica monolith contained within the DNA extraction chamber and the microfluidic device sealed using polymer electrodes. The washing and elution steps for DNA extraction were carried out using EOP, resulting in transfer of the eluted DNA into the PCR chamber. Thermal cycling, achieved using a Peltier element, resulted in amplification of the Amelogenin locus as confirmed using conventional capillary gel electrophoresis. It was demonstrated that the PCR reagents could be stored in the microfluidic device for at least 8 weeks at 4 °C with no significant loss of activity. Such methodology lends itself to the production of 'ready-to-use' microfluidic devices containing all the necessary reagents for sample processing, with many obvious applications in forensics and clinical medicine.

  20. Rapid Extraction of Genomic DNA from Medically Important Yeasts and Filamentous Fungi by High-Speed Cell Disruption

    OpenAIRE

    Müller, Frank-Michael C.; Werner, Katherine E.; Kasai, Miki; Francesconi, Andrea; Chanock, Stephen J.; Walsh, Thomas J.

    1998-01-01

    Current methods of DNA extraction from different fungal pathogens are often time-consuming and require the use of toxic chemicals. DNA isolation from some fungal organisms is difficult due to cell walls or capsules that are not readily susceptible to lysis. We therefore investigated a new and rapid DNA isolation method using high-speed cell disruption (HSCD) incorporating chaotropic reagents and lysing matrices in comparison to standard phenol-chloroform (PC) extraction protocols for isolatio...

  1. Optimal DNA Isolation Method for Detection of Nontuberculous Mycobacteria by Polymerase Chain Reaction.

    Science.gov (United States)

    Mohammadi, Samira; Esfahani, Bahram Nasr; Moghim, Sharareh; Mirhendi, Hossein; Zaniani, Fatemeh Riyahi; Safaei, Hajieh Ghasemian; Fazeli, Hossein; Salehi, Mahshid

    2017-01-01

    Nontuberculous mycobacteria (NTM) are a group of opportunistic pathogens and these are widely dispersed in water and soil resources. Identification of mycobacteria isolates by conventional methods including biochemical tests, growth rates, colony pigmentation, and presence of acid-fast bacilli is widely used, but these methods are time-consuming, labor-intensive, and may sometimes remain inconclusive. The DNA was extracted from NTM cultures using CTAB, Chelex, Chelex + Nonidet P-40, FTA ® Elute card, and boiling The quantity and quality of the DNA extracted via these methods were determined using UV-photometer at 260 and 280 nm, and polymerase chain reaction (PCR) amplification of the heat-shock protein 65 gene with serially diluted DNA samples. The CTAB method showed more positive results at 1:10-1:100,000 at which the DNA amount was substantial. With the Chelex method of DNA extraction, PCR amplification was detected at 1:10 and 1:1000 dilutions. According to the electrophoresis results, the CTAB and Chelex DNA extraction methods were more successful in comparison with the others as regard producing suitable concentrations of DNA with the minimum use of PCR inhibitor.

  2. Optimal DNA Isolation Method for Detection of Nontuberculous Mycobacteria by Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Samira Mohammadi

    2017-01-01

    Full Text Available Background: Nontuberculous mycobacteria (NTM are a group of opportunistic pathogens and these are widely dispersed in water and soil resources. Identification of mycobacteria isolates by conventional methods including biochemical tests, growth rates, colony pigmentation, and presence of acid-fast bacilli is widely used, but these methods are time-consuming, labor-intensive, and may sometimes remain inconclusive. Materials and Methods: The DNA was extracted from NTM cultures using CTAB, Chelex, Chelex + Nonidet P-40, FTA® Elute card, and boiling The quantity and quality of the DNA extracted via these methods were determined using UV-photometer at 260 and 280 nm, and polymerase chain reaction (PCR amplification of the heat-shock protein 65 gene with serially diluted DNA samples. Results: The CTAB method showed more positive results at 1:10–1:100,000 at which the DNA amount was substantial. With the Chelex method of DNA extraction, PCR amplification was detected at 1:10 and 1:1000 dilutions. Conclusions: According to the electrophoresis results, the CTAB and Chelex DNA extraction methods were more successful in comparison with the others as regard producing suitable concentrations of DNA with the minimum use of PCR inhibitor.

  3. Batch experiments versus soil pore water extraction--what makes the difference in isoproturon (bio-)availability?

    Science.gov (United States)

    Folberth, Christian; Suhadolc, Metka; Scherb, Hagen; Munch, Jean Charles; Schroll, Reiner

    2009-10-01

    Two approaches to determine pesticide (bio-)availability in soils (i) batch experiments with "extraction with an excess of water" (EEW) and (ii) the recently introduced "soil pore water (PW) extraction" of pesticide incubated soil samples have been compared with regard to the sorption behavior of the model compound isoproturon in soils. A significant correlation between TOC and adsorbed pesticide amount was found when using the EEW approach. In contrast, there was no correlation between TOC and adsorbed isoproturon when using the in situ PW extraction method. Furthermore, sorption was higher at all concentrations in the EEW method when comparing the distribution coefficients (K(d)) for both methods. Over all, sorption in incubated soil samples at an identical water tension (-15 kPa) and soil density (1.3 g cm(-3)) appears to be controlled by a complex combination of sorption driving soil parameters. Isoproturon bioavailability was found to be governed in different soils by binding strength and availability of sorption sites as well as water content, whereas the dominance of either one of these factors seems to depend on the individual composition and characteristics of the respective soil sample. Using multiple linear regression analysis we obtained furthermore indications that the soil pore structure is affected by the EEW method due to disaggregation, resulting in a higher availability of pesticide sorption sites than in undisturbed soil samples. Therefore, it can be concluded that isoproturon sorption is overestimated when using the EEW method, which should be taken into account when using data from this approach or similar batch techniques for risk assessment analysis.

  4. Does DNA extraction affect the physical and chemical composition of historical cod (Gadus morhua) otoliths?

    DEFF Research Database (Denmark)

    Therkildsen, Nina Overgaard; Eg Nielsen, Einar; Hüssy, Karin

    2010-01-01

    Archived otoliths constitute an important source of historical DNA for use in temporal genetic studies, but such otoliths are also valuable for other research applications, e.g. growth or microchemistry studies, where information about the past is of relevance. Consequently, there are potentially...... conflicting interests regarding how the limited and irreplaceable otolith collections should be used. To resolve this, it is important to find out whether DNA extraction damages otoliths such that they can no longer be used for other research purposes or whether individual otoliths can be used in multiple...... applications. We examined the effects of three different DNA extraction methods on the elemental composition, the morphology, and the clarity of annual growth increments for successful age estimation of Atlantic cod (Gadus morhua) otoliths that had been archived for 0–31 years. The three extraction methods...

  5. Critical points of DNA quantification by real-time PCR--effects of DNA extraction method and sample matrix on quantification of genetically modified organisms.

    Science.gov (United States)

    Cankar, Katarina; Stebih, Dejan; Dreo, Tanja; Zel, Jana; Gruden, Kristina

    2006-08-14

    Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to

  6. Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification.

    Science.gov (United States)

    Yang, Qi; Franco, Christopher M M; Zhang, Wei

    2015-10-01

    Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined