Quantitative detection of chloroethene-reductive bacteria Dehalococcoides spp. using alternately binding probe competitive polymerase chain reaction

Ryo Miyata, Ken Adachi, Hidenori Tani, Shinya Kurata, Kazunori Nakamura, Satoshi Tsuneda, Yuji Sekiguchi, Naohiro Noda

    Research output: Contribution to journalArticle

    5 Citations (Scopus)

    Abstract

    Dehalococcoides spp. are responsible for the reductive dehalogenation of environmental contaminants and are candidates for engineered bioremediation. The development of a sensitive, reliable, and rapid method for the quantification of Dehalococcoides spp. is required for the effective use of the organisms in bioremediation sites. Here, we describe the quantification of the 16S rRNA gene of Dehalococcoides spp. using a recently developed quantification method named alternately binding probe competitive PCR (ABC-PCR). The primers and probe sets that were newly designed for ABC-PCR were found to have a high specificity for Dehalococcoides spp. The standard curve of ABC-PCR had a good fitting (R = 0.999), and the lower detection limit was 10 copies/μl of template DNA. We also investigated the effects of inherent PCR-inhibiting compounds in an environmental sample on the quantification using ABC-PCR or real-time PCR by adding the soil extraction solution to PCR mixtures. ABC-PCR was more robust against the PCR amplification inhibitors than real-time PCR. The copy number of the 16S rRNA gene of Dehalococcoides spp. in soil and groundwater samples was successfully quantified using ABC-PCR. In conclusion, ABC-PCR is useful for the quantification of Dehalococcoides spp. populations and dynamics at bioremediation sites.

    Original languageEnglish
    Pages (from-to)131-137
    Number of pages7
    JournalMolecular and Cellular Probes
    Volume24
    Issue number3
    DOIs
    Publication statusPublished - 2010 Jun

    Fingerprint

    Competitive Binding
    Bacteria
    Polymerase Chain Reaction
    Environmental Biodegradation
    rRNA Genes
    Real-Time Polymerase Chain Reaction
    Soil
    Groundwater
    Population Dynamics
    Limit of Detection

    Keywords

    • Alternately binding probe competitive PCR
    • Bioremediation
    • Dehalococcoides
    • DNA quantification
    • Fluorescence quenching

    ASJC Scopus subject areas

    • Cell Biology
    • Molecular Biology

    Cite this

    Quantitative detection of chloroethene-reductive bacteria Dehalococcoides spp. using alternately binding probe competitive polymerase chain reaction. / Miyata, Ryo; Adachi, Ken; Tani, Hidenori; Kurata, Shinya; Nakamura, Kazunori; Tsuneda, Satoshi; Sekiguchi, Yuji; Noda, Naohiro.

    In: Molecular and Cellular Probes, Vol. 24, No. 3, 06.2010, p. 131-137.

    Research output: Contribution to journalArticle

    Miyata, Ryo ; Adachi, Ken ; Tani, Hidenori ; Kurata, Shinya ; Nakamura, Kazunori ; Tsuneda, Satoshi ; Sekiguchi, Yuji ; Noda, Naohiro. / Quantitative detection of chloroethene-reductive bacteria Dehalococcoides spp. using alternately binding probe competitive polymerase chain reaction. In: Molecular and Cellular Probes. 2010 ; Vol. 24, No. 3. pp. 131-137.
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