TY - JOUR
T1 - Rapid and sensitive identification of marine bacteria by an improved in situ DNA hybridization chain reaction (quickHCR-FISH)
AU - Yamaguchi, Tsuyoshi
AU - Fuchs, Bernhard Maximilian
AU - Amann, Rudolf
AU - Kawakami, Shuji
AU - Kubota, Kengo
AU - Hatamoto, Masashi
AU - Yamaguchi, Takashi
N1 - Funding Information:
We thank Jörg Wulf from the Max Planck Institute for Marine Microbiology for technical assistance. David Probandt and Stefan Dyksma kindly provided the marine sediment samples used in this study. The study was supported by the Max Planck Society and Japan Society for the Promotion of Science (JSPS) . Tsuyoshi Yamaguchi was supported by a JSPS Research Fellowship for Young Scientists.
Publisher Copyright:
© 2015 Elsevier GmbH.
Copyright:
Copyright 2016 Elsevier B.V., All rights reserved.
PY - 2015/9/1
Y1 - 2015/9/1
N2 - 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 2 h 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 3 h. Thus, this newly developed protocol could be an attractive alternative to CARD-FISH for the detection and visualization of microorganisms in their environmental context.
AB - 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 2 h 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 3 h. Thus, this newly developed protocol could be an attractive alternative to CARD-FISH for the detection and visualization of microorganisms in their environmental context.
KW - In situ DNA-HCR
KW - Marine bacteria
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U2 - 10.1016/j.syapm.2015.06.007
DO - 10.1016/j.syapm.2015.06.007
M3 - Article
C2 - 26215142
AN - SCOPUS:84966587130
VL - 38
SP - 400
EP - 405
JO - Systematic and Applied Microbiology
JF - Systematic and Applied Microbiology
SN - 0723-2020
IS - 6
ER -
