Detection of  Clostridium botulinum  group III in environmental samples from farms by real-time PCR using four commercial DNA extraction kits

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Abstract

Objectives

Few studies have tested DNA extraction methods to optimize the detection of Clostridium botulinum in environmental samples that can be collected during animal botulism outbreaks. In this study, we evaluated four commercial DNA extraction kits for the detection of C. botulinum group III in 82 various environmental samples (9 manure, 53 swabs, 3 insects, 8 water, 1 silage and 8 soil samples) collected in a context of animal botulism outbreaks.

Results

The PowerSoil ^® kit was the most efficient for almost all matrices (83.6% of the 73 tested samples), except manure for which the NucleoSpin ^® Soil kit was the most efficient. The NucleoSpin ^® Soil kit enabled detection in 75.3%, the QIAamp ^® DNA Mini Kit in 68.5%, and the QIAamp ^® Fast DNA Stool Mini Kit in 45.2%. However, the NucleoSpin ^® Soil kit detected C. botulinum in 9 of the 9 manure samples tested, while the PowerSoil ^® kit found C. botulinum in only two samples, and the other two kits in none of the samples. This study showed that PowerSoil ^® can be recommended for DNA extraction from environmental samples except for manure, for which the NucleoSpin ^® Soil kit appeared to be far more appropriate.

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Most cited references 17

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Biases during DNA extraction of activated sludge samples revealed by high throughput sequencing

Feng Guo, Tong Zhang (2012)

Standardization of DNA extraction is a fundamental issue of fidelity and comparability in investigations of environmental microbial communities. Commercial kits for soil or feces are often adopted for studies of activated sludge because of a lack of specific kits, but they have never been evaluated regarding their effectiveness and potential biases based on high throughput sequencing. In this study, seven common DNA extraction kits were evaluated, based on not only yield/purity but also sequencing results, using two activated sludge samples (two sub-samples each, i.e. ethanol-fixed and fresh, as-is). The results indicate that the bead-beating step is necessary for DNA extraction from activated sludge. The two kits without the bead-beating step yielded very low amounts of DNA, and the least abundant operational taxonomic units (OTUs), and significantly underestimated the Gram-positive Actinobacteria, Nitrospirae, Chloroflexi, and Alphaproteobacteria and overestimated Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, and the rare phyla whose cell walls might have been readily broken. Among the other five kits, FastDNA@ SPIN Kit for Soil extracted the most and the purest DNA. Although the number of total OTUs obtained using this kit was not the highest, the abundant OTUs and abundance of Actinobacteria demonstrated its efficiency. The three MoBio kits and one ZR kit produced fair results, but had a relatively low DNA yield and/or less Actinobacteria-related sequences. Moreover, the 50 % ethanol fixation increased the DNA yield, but did not change the sequenced microbial community in a significant way. Based on the present study, the FastDNA SPIN kit for Soil is recommended for DNA extraction of activated sludge samples. More importantly, the selection of the DNA extraction kit must be done carefully if the samples contain dominant lysing-resistant groups, such as Actinobacteria and Nitrospirae. Electronic supplementary material The online version of this article (doi:10.1007/s00253-012-4244-4) contains supplementary material, which is available to authorized users.

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Fecal microbiota analysis: an overview of sample collection methods and sequencing strategies.

Vincent Thomas, James Clark, Joël Doré (2015)

Despite huge interest, there are still no universally accepted standards to conduct clinical studies in the field of gut microbiota analysis. Stool material is frequently used as a proxy of gut microbiota, but many different protocols can be used for collection and DNA extraction. Whereas 16S rRNA encoding gene amplification and sequencing has been widely used to study the composition of bacterial populations, it is now being challenged by the random, shotgun approach that brings far more information, although at a higher cost. In this review we give an overview of existing methods and important points to consider when conducting gut microbiota studies, with the objective to provide recommendations to those who would like to conduct such research.

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An evaluation of commercial DNA extraction kits for the isolation of bacterial spore DNA from soil.

Paige D. Anders, R Aranda, Elizabeth J. Robertson … (2010)

To evaluate six commercial DNA extraction kits for their ability to isolate PCR-quality DNA from Bacillus spores in various soil samples. Three soils were inoculated with various amounts of Bacillus cereus spores to simulate an outbreak or intentional release of the threat agent Bacillus anthracis. DNA was isolated from soil samples using six commercial DNA extraction kits. Extraction and purification efficiencies were assessed using a duplex real-time PCR assay that included an internal positive control. The FastDNA(®) SPIN kit for Soil showed the highest DNA extraction yield, while the E.Z.N.A.(®) Soil DNA and PowerSoil(®) DNA Isolation kits showed the highest efficiencies in removing PCR inhibitors from loam soil extracts. The results of this study suggest that commercially available extraction kits can be used to extract PCR-quality DNA from bacterial spores in soil. The selection of an appropriate extraction kit should depend on the characteristics of the soil sample and the intended downstream application. The results of this study aid in the selection of an appropriate DNA extraction kit for a given soil sample. Its application could expedite sample processing for real-time PCR detection of a pathogen in soil. © 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.

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Author and article information

Contributors

Caroline Le Maréchal: +33 2 96 01 85 33 , caroline.lemarechal@anses.fr

Sarah Fourour: sarah.fourour@anses.fr

Valentine Ballan: valentine.ballan@gmail.com

Sandra Rouxel: Sandra.ROUXEL@anses.fr

Rozenn Souillard: Rozenn.SOUILLARD@anses.fr

Marianne Chemaly: Marianne.CHEMALY@anses.fr

Journal

Journal ID (nlm-ta): BMC Res Notes

Journal ID (iso-abbrev): BMC Res Notes

Title: BMC Research Notes

Publisher: BioMed Central (London )

ISSN (Electronic): 1756-0500

Publication date (Electronic): 4 July 2018

Publication date PMC-release: 4 July 2018

Publication date Collection: 2018

Volume: 11

Electronic Location Identifier: 441

Affiliations

[1 ]ANSES, Laboratoire de Ploufragan-Plouzané, Unité Hygiène et Qualité des Produits Avicoles et Porcins, Université Bretagne-Loire, BP 53, 22440 Ploufragan, France

[2 ]ANSES, Laboratoire de Ploufragan-Plouzané, Unité d’Épidémiologie et Bien-être en Aviculture et Cuniculture, Université Bretagne-Loire, BP 53, 22440 Ploufragan, France

Article

Publisher ID: 3549

DOI: 10.1186/s13104-018-3549-5

PMC ID: 6030735

PubMed ID: 29973253

SO-VID: 18471248-a450-4c92-a900-019787c59662

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 16 May 2018

Date accepted : 27 June 2018

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100003199, France AgriMer;

Award ID: BOTUNET

Award Recipient : Caroline Le Maréchal

Funded by: DGAl

Funded by: Conseil général des Côtes d'Armor

Custom metadata

ScienceOpen disciplines: Medicine

Keywords: clostridium botulinum,dna extraction,real-time pcr,detection,freezing

Data availability:

ScienceOpen disciplines: Medicine

Keywords: clostridium botulinum, dna extraction, real-time pcr, detection, freezing

Detection of Clostridium botulinum group III in environmental samples from farms by real-time PCR using four commercial DNA extraction kits

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Abstract

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IOP Publishing: Coronavirus

Most cited references 17

Biases during DNA extraction of activated sludge samples revealed by high throughput sequencing

Fecal microbiota analysis: an overview of sample collection methods and sequencing strategies.

An evaluation of commercial DNA extraction kits for the isolation of bacterial spore DNA from soil.

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