title: In situ DNA-hybridization chain reaction (HCR): a facilitated in situ HCR system for the detection of environmental microorganisms.
abstract: In situ detection of microorganisms by fluorescence in situ hybridization (FISH) is a powerful tool for environmental microbiology, but analyses can be hampered by low rRNA content in target organisms, especially in oligotrophic environments. Here, we present a non-enzymatic, hybridization chain reaction (HCR)-based signal amplified in situ whole-cell detection technique (in situ DNA-HCR). The components of the amplification buffer were optimized to polymerize DNA amplifier probes for in situ DNA-HCR. In situ hybridization of initiator probes followed by signal amplification via HCR produced bright signals with high specificity and probe permeation into cells. The detection rates for Bacteria in a seawater sample and Archaea in anaerobic sludge samples were comparable with or greater than those obtained by catalyzed reporter deposition (CARD)-FISH or standard FISH. Detection of multiple organisms (Bacteria, Archaea and Methanosaetaceae) in an anaerobic sludge sample was achieved by simultaneous in situ DNA-HCR. In summary, in situ DNA-HCR is a simple and easy technique for detecting single microbial cells and enhancing understanding of the ecology and behaviour of environmental microorganisms in situ.
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata, 940-2188, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yamaguchi
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata, 940-2188, Japan.|Department of Construction Systems Engineering, Anan National College of Technology, 265 Aoki Minobayashi, Anan, Tokushima, 774-0017, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Kawakami
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata, 940-2188, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Hatamoto
authors.affiliation: Department of Subsurface Geobiology Analysis and Research (D-SUGAR), Japan Agency for Marine-Earth Science & Technology (JAMSTEC), Yokosuka, Kanagawa, 237-0061, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Imachi
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata, 940-2188, Japan.|Department of Civil and Environmental Engineering, Tohoku University, 6-6-06 Aoba, Sendai, Miyagi, 980-8579, Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Takahashi
authors.affiliation: Department of Civil Engineering, Nagaoka National College of Technology, 888 Nishikatagai, Nagaoka, Niigata, 940-8532, Japan.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Araki
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata, 940-2188, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamaguchi
authors.affiliation: Department of Civil and Environmental Engineering, Tohoku University, 6-6-06 Aoba, Sendai, Miyagi, 980-8579, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Kubota
mesh_terms: D001105:Archaea / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D019641:DNA, Archaeal / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004783:Environmental Microbiology; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D019640:Methanosarcinales / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D015345:Oligonucleotide Probes / Q000235:genetics; D012623:Seawater / Q000382:microbiology; D012680:Sensitivity and Specificity; D012722:Sewage / Q000382:microbiology
chemical_list: D019641:DNA, Archaeal; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D012722:Sewage