Quantitative detection of chloroethene-reductive bacteria Dehalococcoides spp. using alternately binding probe competitive Polymerase Chain Reaction.
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/microl 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.
Miyata
Ryo
R
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, Tsukuba, Ibaraki, Japan.
Adachi
Ken
K
Tani
Hidenori
H
Kurata
Shinya
S
Nakamura
Kazunori
K
Tsuneda
Satoshi
S
Sekiguchi
Yuji
Y
Noda
Naohiro
N
D001673:Biodegradation, Environmental; D041862:Chloroflexi / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D015342:DNA Probes / Q000235:genetics*; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012988:Soil Microbiology; D013045:Species Specificity; D014752:Vinyl Chloride / Q000378:metabolism*; D014871:Water Microbiology
D015342:DNA Probes; D012336:RNA, Ribosomal, 16S; D014752:Vinyl Chloride