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Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR.

Abstract

AIMS

The microbiota of the human intestinal tract constitutes a complex ecosystem. We report the design and optimization of an extensive set of 16S rDNA-targeted species- and group-specific primers for more accurate quantification of bacteria from faecal samples with real-time PCR.

METHODS AND RESULTS

A linear range of quantification between 0.1-10 pg and 10 ng of specific target genome was obtained, which corresponds to detection of ca 30-4500 to 1.9 x 10(6)-6.0 x 10(6) target bacterial genomes. Functionality of the assays was confirmed by quantification of target bacterial DNA from faecal DNA preparations of healthy volunteers and irritable bowel syndrome (IBS) patients. Additionally, spiking of faecal preparations with Helicobacter pylori, Clostridium difficile or Campylobacter jejuni was used to confirm the accurate and sensitive quantification.

CONCLUSIONS

Real-time PCR is a very sensitive and precise technique for an extensive quantitative evaluation of gut microbiota and is feasible for detection of human pathogens from faecal samples.

SIGNIFICANCE AND IMPACT OF THE STUDY

To design and optimize an extensive set of real-time PCR assays targeting a large group of predominant and pathogenic GI microbial species for further use in updating the current knowledge of the putative role of gut microbiota in health and disease.

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  • Authors+Show Affiliations

    ,

    Section of Microbiology, Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, Helsinki University, Helsinki, Finland.

    , , ,

    Source

    Journal of applied microbiology 97:6 2004 pg 1166-77

    MeSH

    Adult
    Bacteria
    Base Sequence
    Clostridium
    DNA Primers
    DNA, Bacterial
    DNA, Ribosomal
    Escherichia coli
    Feces
    Fluorescent Dyes
    Genome, Bacterial
    Helicobacter pylori
    Humans
    Irritable Bowel Syndrome
    Organic Chemicals
    Polymerase Chain Reaction
    Species Specificity

    Pub Type(s)

    Journal Article
    Research Support, Non-U.S. Gov't

    Language

    eng

    PubMed ID

    15546407

    Citation

    Rinttilä, T, et al. "Development of an Extensive Set of 16S rDNA-targeted Primers for Quantification of Pathogenic and Indigenous Bacteria in Faecal Samples By Real-time PCR." Journal of Applied Microbiology, vol. 97, no. 6, 2004, pp. 1166-77.
    Rinttilä T, Kassinen A, Malinen E, et al. Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR. J Appl Microbiol. 2004;97(6):1166-77.
    Rinttilä, T., Kassinen, A., Malinen, E., Krogius, L., & Palva, A. (2004). Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR. Journal of Applied Microbiology, 97(6), pp. 1166-77.
    Rinttilä T, et al. Development of an Extensive Set of 16S rDNA-targeted Primers for Quantification of Pathogenic and Indigenous Bacteria in Faecal Samples By Real-time PCR. J Appl Microbiol. 2004;97(6):1166-77. PubMed PMID: 15546407.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR. AU - Rinttilä,T, AU - Kassinen,A, AU - Malinen,E, AU - Krogius,L, AU - Palva,A, PY - 2004/11/18/pubmed PY - 2005/4/12/medline PY - 2004/11/18/entrez SP - 1166 EP - 77 JF - Journal of applied microbiology JO - J. Appl. Microbiol. VL - 97 IS - 6 N2 - AIMS: The microbiota of the human intestinal tract constitutes a complex ecosystem. We report the design and optimization of an extensive set of 16S rDNA-targeted species- and group-specific primers for more accurate quantification of bacteria from faecal samples with real-time PCR. METHODS AND RESULTS: A linear range of quantification between 0.1-10 pg and 10 ng of specific target genome was obtained, which corresponds to detection of ca 30-4500 to 1.9 x 10(6)-6.0 x 10(6) target bacterial genomes. Functionality of the assays was confirmed by quantification of target bacterial DNA from faecal DNA preparations of healthy volunteers and irritable bowel syndrome (IBS) patients. Additionally, spiking of faecal preparations with Helicobacter pylori, Clostridium difficile or Campylobacter jejuni was used to confirm the accurate and sensitive quantification. CONCLUSIONS: Real-time PCR is a very sensitive and precise technique for an extensive quantitative evaluation of gut microbiota and is feasible for detection of human pathogens from faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: To design and optimize an extensive set of real-time PCR assays targeting a large group of predominant and pathogenic GI microbial species for further use in updating the current knowledge of the putative role of gut microbiota in health and disease. SN - 1364-5072 UR - https://www.unboundmedicine.com/medline/citation/15546407/Development_of_an_extensive_set_of_16S_rDNA_targeted_primers_for_quantification_of_pathogenic_and_indigenous_bacteria_in_faecal_samples_by_real_time_PCR_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=1364-5072&date=2004&volume=97&issue=6&spage=1166 DB - PRIME DP - Unbound Medicine ER -