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Muriel Vayssier-taussat - One of the best experts on this subject based on the ideXlab platform.

  • Determination of an efficient and reliable method for DNA Extraction from ticks
    Veterinary research, 2004
    Co-Authors: Lénaïg Halos, Taoufik Jamal, Laurence Vial, Renaud Maillard, Antonia Suau, Arnaud Le Menach, Henri-jean Boulouis, Muriel Vayssier-taussat
    Abstract:

    Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, Extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA Extraction protocols based on an enzymatic digestion by proteinase K followed by DNA Extraction by a commercial kit (method 1), or on mortar crushing, proteinase K digestion and phenol/chloroform DNA Extraction (method 2) and fine crushing with a beads beater, proteinase K digestion and DNA Extraction using a commercial kit (method 3). The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the tick mitochondrial 16S rRNA gene using tick-specific primers. With method 1, 23/30 (77%) of the samples were extracted; with method 2, 30/31 (97%) of the samples were extracted and with method 3, 30/30 (100%) of the samples were extracted. DNA Extraction efficiency using method 3 is significantly higher than DNA Extraction efficiency using method 1 (100% versus 77%, P < 0.05). There was no significant difference between methods 2 and 3. Method 3 was however more adapted to cohort studies than method 2. This technique was validated for cohort tick DNA Extraction and applicable to the treatment of small samples such as nymphs and soft ticks with 100% efficiency. tick / DNA Extraction / PCR / nymphal stage

  • Determination of an efficient and reliable method for DNA Extraction from ticks
    Veterinary Research, 2004
    Co-Authors: Lénaïg Halos, Taoufik Jamal, Laurence Vial, Renaud Maillard, Antonia Suau, Arnaud Le Menach, Henri-jean Boulouis, Muriel Vayssier-taussat
    Abstract:

    Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, Extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA Extraction protocols based on an enzymatic digestion by proteinase K followed by DNA Extraction by a commercial kit (method 1), or on mortar crushing, proteinase K digestion and phenol/chloroform DNA Extraction (method 2) and fine crushing with a beads beater, proteinase K digestion and DNA Extraction using a commercial kit (method 3). The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the tick mitochondrial 16S rRNA gene using tick-specific primers. With method 1, 23/30 (77%) of the samples were extracted; with method 2, 30/31 (97%) of the samples were extracted and with method 3, 30/30 (100%) of the samples were extracted. DNA Extraction efficiency using method 3 is significantly higher than DNA Extraction efficiency using method 1 (100% versus 77%, P < 0.05). There was no significant difference between methods 2 and 3. Method 3 was however more adapted to cohort studies than method 2. This technique was validated for cohort tick DNA Extraction and applicable to the treatment of small samples such as nymphs and soft ticks with 100% efficiency.

Michael Hofreiter - One of the best experts on this subject based on the ideXlab platform.

  • Nondestructive DNA Extraction from Museum Specimens
    Methods in molecular biology (Clifton N.J.), 2011
    Co-Authors: Michael Hofreiter
    Abstract:

    Natural history museums around the world hold millions of animal and plant specimens that are potentially amenable to genetic analyses. With more and more populations and species becoming extinct, the importance of these specimens for phylogenetic and phylogeographic analyses is rapidly increasing. However, as most DNA Extraction methods damage the specimens, nondestructive Extraction methods are useful to balance the demands of molecular biologists, morphologists, and museum curators. Here, I describe a method for nondestructive DNA Extraction from bony specimens (i.e., bones and teeth). In this method, the specimens are soaked in Extraction buffer, and DNA is then purified from the soaking solution using adsorption to silica. The method reliably yields mitochondrial and often also nuclear DNA. The method has been adapted to DNA Extraction from other types of specimens such as arthropods.

Lénaïg Halos - One of the best experts on this subject based on the ideXlab platform.

  • Determination of an efficient and reliable method for DNA Extraction from ticks
    Veterinary research, 2004
    Co-Authors: Lénaïg Halos, Taoufik Jamal, Laurence Vial, Renaud Maillard, Antonia Suau, Arnaud Le Menach, Henri-jean Boulouis, Muriel Vayssier-taussat
    Abstract:

    Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, Extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA Extraction protocols based on an enzymatic digestion by proteinase K followed by DNA Extraction by a commercial kit (method 1), or on mortar crushing, proteinase K digestion and phenol/chloroform DNA Extraction (method 2) and fine crushing with a beads beater, proteinase K digestion and DNA Extraction using a commercial kit (method 3). The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the tick mitochondrial 16S rRNA gene using tick-specific primers. With method 1, 23/30 (77%) of the samples were extracted; with method 2, 30/31 (97%) of the samples were extracted and with method 3, 30/30 (100%) of the samples were extracted. DNA Extraction efficiency using method 3 is significantly higher than DNA Extraction efficiency using method 1 (100% versus 77%, P < 0.05). There was no significant difference between methods 2 and 3. Method 3 was however more adapted to cohort studies than method 2. This technique was validated for cohort tick DNA Extraction and applicable to the treatment of small samples such as nymphs and soft ticks with 100% efficiency. tick / DNA Extraction / PCR / nymphal stage

  • Determination of an efficient and reliable method for DNA Extraction from ticks
    Veterinary Research, 2004
    Co-Authors: Lénaïg Halos, Taoufik Jamal, Laurence Vial, Renaud Maillard, Antonia Suau, Arnaud Le Menach, Henri-jean Boulouis, Muriel Vayssier-taussat
    Abstract:

    Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, Extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA Extraction protocols based on an enzymatic digestion by proteinase K followed by DNA Extraction by a commercial kit (method 1), or on mortar crushing, proteinase K digestion and phenol/chloroform DNA Extraction (method 2) and fine crushing with a beads beater, proteinase K digestion and DNA Extraction using a commercial kit (method 3). The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the tick mitochondrial 16S rRNA gene using tick-specific primers. With method 1, 23/30 (77%) of the samples were extracted; with method 2, 30/31 (97%) of the samples were extracted and with method 3, 30/30 (100%) of the samples were extracted. DNA Extraction efficiency using method 3 is significantly higher than DNA Extraction efficiency using method 1 (100% versus 77%, P < 0.05). There was no significant difference between methods 2 and 3. Method 3 was however more adapted to cohort studies than method 2. This technique was validated for cohort tick DNA Extraction and applicable to the treatment of small samples such as nymphs and soft ticks with 100% efficiency.

Michael Schloter - One of the best experts on this subject based on the ideXlab platform.

  • Soil microbial diversity : an ISO standard for soil DNA Extraction
    Journal of Soils and Sediments, 2010
    Co-Authors: Laurent Philippot, Cristina Abbate, Antonio Bispo, Thierry Chesnot, Sara Hallin, Philippe Lemanceau, Kristina Lindstrom, Pascal Pandard, Esperanza Romero, Michael Schloter
    Abstract:

    Soil carries out functions that are crucial for the environment and life on earth and is therefore an essential non-renewable resource for mankind. Recently, the European Soil Framework Directive proposal (COM(2006)232) indicated that soil is under increasing environmental pressure mostly due to the intensification of human activities, which are damaging the capacity of soil to continue to perform in full its broad variety of crucial functions. Most of these soil functions are dependent on microorganisms inhabiting the soil. The diversity of soil microorganisms is the highest on earth with estimates of several thousand to several million different genomes per gram of soil (Whitman et al. 1998; Torsvik et al. 2002; Zhang and Xu 2008). However, fundamental understanding of the diversity and ecology of microbial communities carrying out soil functions has been hampered by our inability to grow most microorganisms under laboratory conditions. Since the early eighties, direct DNA-based methods have been developed to circumvent the biases resulting from the low number of microorganisms that could be cultured, thus hampering our understanding of microbial diversity in soil (Torsvik 1980; Porteous et al. 1991; Tsai et al. 1991; Smalla et al. 1993; Zhou et al. 1996; He et al. 2005). These methods were based on direct Extraction of the DNA from micro-organisms living in the soil, using various lysis treatments. Since then, numerous articles have been published describing either new or improved methods for soil DNA Extraction and at least ten companies are commercializing “Soil DNA” Extraction kits. The on-going success of these direct molecular methods for studying soil micro-organisms is reflected by the fact that more than 1,000 articles are now published yearly using some type of soil DNA Extraction method. Unfortunately, the wide use of these methods has resulted in a huge number of laboratory or even user-specific protocols, which contain minor to major modifications of the existing methods or kits. However, the choice of DNA Extraction method is far from being inconsequential as it affects the picture of the microbial diversity present in a soil sample (Frostegard et al. 1999; Krsek et al. 1999; Martin-Laurent et al. 2001; He et al. 2005). To tackle this crucial problem and ensure comparable data, a standard soil DNA Extraction method should be used. This is becoming all the more important because studies of soil microbial diversity based on soil DNA Extraction are generating an exponential amount of sequence data thanks to rapid advances in sequencing technologies. For this purpose, we proposed in 2006 the standardization of soil DNA Extraction to the International Organization for Standardization (ISO), with support from the French Standards Association and the French Environment and Energy Management Agency. The need for an international standard for soil DNA Extraction has been rapidly recognized by ISO members as a priority and formally agreed upon. The ISO 11063 Soil quality method to directly extract DNA from soil samples is now being prepared by the Technical Committee ISO/TC 190, Subcommittee SC4, Biological methods. The standard was developed based on the publication made by Martin-Laurent et al. (2001). In the first phase, the technical scope of the future standard has been defined by a working group, consisting of expert researchers from countries interested in the subject matter. It was decided that since DNA purification is dependent on soil type, only the actual DNA Extraction step, which is likely to generate the main differences between methods, would be subject to standardization. After a successful French inter-laboratory assay, we were requested during the ISO/TC 190 meeting in Delft in November 2008 to set up an international ring test for a final validation of the method. This task involved ten different laboratories from seven countries. Six soils collected in different European Countries were chosen for this study. The final report of this ring test was included in the revised of the standard and sent to the ISO in January 2010. After a 5 months ballot, more than two thirds of the votes cast by the national standards bodies members were positive and the ISO standard for Soil DNA Extraction has been approved. The normative document will now be disseminated within the scientific community. However, adoption of the ISO standard is voluntary and both information and international cooperation will be needed for the acceptance and success of ISO/CD 11063 standard. We hope that this standardization of a soil DNA Extraction method will be an important first step towards the unification of soil microbiologists.

Henri-jean Boulouis - One of the best experts on this subject based on the ideXlab platform.

  • Determination of an efficient and reliable method for DNA Extraction from ticks
    Veterinary research, 2004
    Co-Authors: Lénaïg Halos, Taoufik Jamal, Laurence Vial, Renaud Maillard, Antonia Suau, Arnaud Le Menach, Henri-jean Boulouis, Muriel Vayssier-taussat
    Abstract:

    Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, Extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA Extraction protocols based on an enzymatic digestion by proteinase K followed by DNA Extraction by a commercial kit (method 1), or on mortar crushing, proteinase K digestion and phenol/chloroform DNA Extraction (method 2) and fine crushing with a beads beater, proteinase K digestion and DNA Extraction using a commercial kit (method 3). The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the tick mitochondrial 16S rRNA gene using tick-specific primers. With method 1, 23/30 (77%) of the samples were extracted; with method 2, 30/31 (97%) of the samples were extracted and with method 3, 30/30 (100%) of the samples were extracted. DNA Extraction efficiency using method 3 is significantly higher than DNA Extraction efficiency using method 1 (100% versus 77%, P < 0.05). There was no significant difference between methods 2 and 3. Method 3 was however more adapted to cohort studies than method 2. This technique was validated for cohort tick DNA Extraction and applicable to the treatment of small samples such as nymphs and soft ticks with 100% efficiency. tick / DNA Extraction / PCR / nymphal stage

  • Determination of an efficient and reliable method for DNA Extraction from ticks
    Veterinary Research, 2004
    Co-Authors: Lénaïg Halos, Taoufik Jamal, Laurence Vial, Renaud Maillard, Antonia Suau, Arnaud Le Menach, Henri-jean Boulouis, Muriel Vayssier-taussat
    Abstract:

    Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, Extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA Extraction protocols based on an enzymatic digestion by proteinase K followed by DNA Extraction by a commercial kit (method 1), or on mortar crushing, proteinase K digestion and phenol/chloroform DNA Extraction (method 2) and fine crushing with a beads beater, proteinase K digestion and DNA Extraction using a commercial kit (method 3). The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the tick mitochondrial 16S rRNA gene using tick-specific primers. With method 1, 23/30 (77%) of the samples were extracted; with method 2, 30/31 (97%) of the samples were extracted and with method 3, 30/30 (100%) of the samples were extracted. DNA Extraction efficiency using method 3 is significantly higher than DNA Extraction efficiency using method 1 (100% versus 77%, P < 0.05). There was no significant difference between methods 2 and 3. Method 3 was however more adapted to cohort studies than method 2. This technique was validated for cohort tick DNA Extraction and applicable to the treatment of small samples such as nymphs and soft ticks with 100% efficiency.