Environmental Microbiology

Volume 17, Issue 7 p. 2532-2541

Research article

In situ DNA-hybridization chain reaction (HCR): a facilitated in situ HCR system for the detection of environmental microorganisms

Tsuyoshi Yamaguchi,

Tsuyoshi Yamaguchi

Department of Environmental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata, 940-2188 Japan

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Shuji Kawakami,

Corresponding Author

Shuji Kawakami

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

For correspondence. E-mail [email protected]; Tel. +81 (884) 23 7127; Fax +81 (884) 23 7127.Search for more papers by this author

Masashi Hatamoto,

Masashi Hatamoto

Department of Environmental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata, 940-2188 Japan

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Hiroyuki Imachi,

Hiroyuki Imachi

Department of Subsurface Geobiology Analysis and Research (D-SUGAR), Japan Agency for Marine-Earth Science & Technology (JAMSTEC), Yokosuka, Kanagawa, 237-0061 Japan

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Masanobu Takahashi,

Masanobu Takahashi

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

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Nobuo Araki,

Nobuo Araki

Department of Civil Engineering, Nagaoka National College of Technology, 888 Nishikatagai, Nagaoka, Niigata, 940-8532 Japan

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Takashi Yamaguchi,

Takashi Yamaguchi

Department of Environmental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata, 940-2188 Japan

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Kengo Kubota,

Kengo Kubota

Department of Civil and Environmental Engineering, Tohoku University, 6-6-06 Aoba, Sendai, Miyagi, 980-8579 Japan

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Tsuyoshi Yamaguchi,

Tsuyoshi Yamaguchi

Department of Environmental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata, 940-2188 Japan

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Shuji Kawakami,

Corresponding Author

Shuji Kawakami

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

For correspondence. E-mail [email protected]; Tel. +81 (884) 23 7127; Fax +81 (884) 23 7127.Search for more papers by this author

Masashi Hatamoto,

Masashi Hatamoto

Department of Environmental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata, 940-2188 Japan

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Hiroyuki Imachi,

Hiroyuki Imachi

Department of Subsurface Geobiology Analysis and Research (D-SUGAR), Japan Agency for Marine-Earth Science & Technology (JAMSTEC), Yokosuka, Kanagawa, 237-0061 Japan

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Masanobu Takahashi,

Masanobu Takahashi

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

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Nobuo Araki,

Nobuo Araki

Department of Civil Engineering, Nagaoka National College of Technology, 888 Nishikatagai, Nagaoka, Niigata, 940-8532 Japan

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Takashi Yamaguchi,

Takashi Yamaguchi

Department of Environmental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata, 940-2188 Japan

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Kengo Kubota,

Kengo Kubota

Department of Civil and Environmental Engineering, Tohoku University, 6-6-06 Aoba, Sendai, Miyagi, 980-8579 Japan

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First published: 19 December 2014

https://doi.org/10.1111/1462-2920.12745

Citations: 58

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Summary

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.

Supporting Information

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emi12745-sup-0001-si.pdf7 MB

Fig. S1. Dissociation curves of the EUB338, CF319a, and Gam42a probes without and with the initiatorH sequence. A: the EUB338 and EUB338-initiatorH probes labeled with AlexaFluor555. B: the CF319a and CF319a-initiatorH probes labeled with Atto550. C: the Gam42a and Gam42a-initiatorH probes labeled with Atto 550. The strains used for drawing dissociation curves were E. coli for EUB338 and Gam42a and Gramella forsetii (DSM17595) for CF319a. Relative intensity indicates the brightness of the probe at respective formamide concentration relative to the fluorescent intensity of the probe at 0% formamide.

Fig. S2. Detection of E. coli cells by in situ DNA-HCR (A–E) or FISH (F-J) at 20% formamide in hybridization buffer. Each double panel depicts DAPI staining (left) and probe staining (right). A: EUB338-initiatorH probe and Cy3-labeled amplifier probes, B: EUB338-1MM-initiatorH probe and Cy3-labeled amplifier probes, C: EUB338-1MM-initiatorH probe + a competitor probe (unlabeled EUB338) and Cy3-labeled amplifier probes, D: EUB338-2MM-initiatorH probe and Cy3-labeled amplifier probes, E: EUB338-3MM-initiatorH probe and Cy3-labeled amplifier probes, F: Cy3-labeled EUB338, G: Cy3-labeled EUB338-IV (single mismatch), H: Cy3-labeled EUB338-IV + a competitor probe (unlabeled EUB338), I: Cy3-labeled EUB338-III (two mismatches), J: Cy3-labeled EUB338-II (three mismatches). The exposure times for in situ DNA-HCR and standard FISH were 33 ms and 100 ms, respectively. The bar represents 10 μm.

Fig. S3. Photomicrographs of EUB338-hybridized marine bacteria after FISH with the Cy3-labeled EUB338 probe (A), CARD-FISH with the HRP-labeled EUB338 probe and tyramide-Cy3 (B), and in situ DNA-HCR with the EUB338-initiatorH probe and the Cy3-labeled amplifier probes (C). Lysozyme treatment was not conducted for all samples. Each double panel depicts DAPI staining (left) and probe staining (right). Exposure times were adjusted for each method. The bar represents 20 μm.

Fig. S4. Photomicrographs of ARC915-hybridized Archaea in anaerobic granular sludge sample after FISH with the Cy3-labeled ARC915 probe (A), CARD-FISH with the HRP-labeled ARC915 probe and tyamide-Cy3 (B), and in situ DNA-HCR with the ARC915-initiatorH probe and the Cy3-labeled amplifier probes (C). Each double panel depicts DAPI staining (left) and probe staining (right). Exposure times were adjusted for each method. The bar represents 20 μm.

Table S1. Components of buffers used for in situ DNA-HCR.

Table S2. DNA probes used for drawing the dissociation curves.

Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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Volume17, Issue7

July 2015

Pages 2532-2541

In situ DNA-hybridization chain reaction (HCR): a facilitated in situ HCR system for the detection of environmental microorganisms

Summary

Supporting Information

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In situ DNA-hybridization chain reaction (HCR): a facilitated in situ HCR system for the detection of environmental microorganisms

Summary

Supporting Information

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