title: Rapid and sensitive PCR-dipstick DNA chromatography for multiplex analysis of the oral microbiota.
abstract: A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces species, and Veillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases.
authors.affiliation: Division of Oral Ecology and Biochemistry, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan ; Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, No. 14, Section 3, South Renmin Road, Chengdu 610041, China.
authors.forename: Lingyang
authors.identifier: 0000-0002-8796-1319
authors.initials: L
authors.lastname: Tian
authors.affiliation: Division of Oral Ecology and Biochemistry, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan.
authors.forename: Takuichi
authors.identifier: 0000-0002-6444-1193
authors.initials: T
authors.lastname: Sato
authors.affiliation: Tohoku University Graduate School of Biomedical Engineering, Sendai 980-8579, Japan ; Future Technology Management Center, Corporate R&D, NGK Insulators, Mizuho, Nagoya 467-8530, Japan.
authors.forename: Kousuke
authors.initials: K
authors.lastname: Niwa
authors.affiliation: Tohoku University Graduate School of Biomedical Engineering, Sendai 980-8579, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Kawase
authors.affiliation: Department of Microbiology, The Forsyth Institute, Cambridge, MA 02142, USA.
authors.forename: Anne C R
authors.initials: AC
authors.lastname: Tanner
authors.affiliation: Division of Oral Ecology and Biochemistry, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan.
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Takahashi
mesh_terms: D000190:Actinomyces / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D003731:Dental Caries / Q000175:diagnosis / Q000235:genetics / Q000382:microbiology*; D003773:Dental Plaque / Q000382:microbiology* / Q000473:pathology; D006801:Humans; D064307:Microbiota / Q000235:genetics*; D009055:Mouth / Q000382:microbiology* / Q000473:pathology; D013295:Streptococcus mutans / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D016986:Streptococcus sobrinus / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D014678:Veillonella / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity