title: Rapid and sensitive identification of marine bacteria by an improved in situ DNA hybridization chain reaction (quickHCR-FISH).
abstract: Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) with rRNA-targeted oligonucleotide probes has significantly improved the identification of microorganisms in various environmental samples. However, one of the major constraints of CARD-FISH is the low probe penetration due to the high molecular weight of the horseradish peroxidase (HRP) label. Recently, this limitation has been overcome by a novel signal amplification approach termed in situ DNA-hybridization chain reaction (in situ DNA-HCR). In this study, we present an improved and accelerated in situ DNA-HCR protocol (quickHCR-FISH) with increased signal intensity, which was approximately 2 times higher than that of standard in situ DNA-HCR. In addition, the amplification time was only 15 min for the extension of amplifier probes from the initiator probe compared to 2h in the original protocol. The quickHCR-FISH was successfully tested for the quantification of marine bacteria with low rRNA contents in both seawater and sediment samples. It was possible to detect the same number of marine bacteria with quickHCR-FISH compared to CARD-FISH within only 3h. Thus, this newly developed protocol could be an attractive alternative to CARD-FISH for the detection and visualization of microorganisms in their environmental context.
authors.affiliation: Max Planck Institute for Marine Microbiology, Celsiusstraße 1, D-28359 Bremen, Germany; Department of Environmental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan. Electronic address: tsuyoshi@stn.nagaokaut.ac.jp.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yamaguchi
authors.affiliation: Max Planck Institute for Marine Microbiology, Celsiusstraße 1, D-28359 Bremen, Germany.
authors.forename: Bernhard Maximilian
authors.initials: BM
authors.lastname: Fuchs
authors.affiliation: Max Planck Institute for Marine Microbiology, Celsiusstraße 1, D-28359 Bremen, Germany.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Amann
authors.affiliation: 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 Civil and Environmental Engineering, Tohoku University, 6-6-06 Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Kubota
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 Environmental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamaguchi
mesh_terms: D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D019015:Geologic Sediments / Q000382:microbiology*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D012623:Seawater / Q000382:microbiology*; D013997:Time Factors
chemical_list: D004269:DNA, Bacterial
keywords: In situ DNA-HCR; Marine bacteria