In situ <scp>DNA‐</scp>hybridization chain reaction (<scp>HCR</scp>): a facilitated in situ <scp>HCR</scp> system for the detection of environmental microorganisms

Yamaguchi, Tsuyoshi; Kawakami, S.; Hatamoto, Masashi; Imachi, Hiroyuki; Takahashi, Masanobu; Araki, Nobuo; Yamaguchi, Takashi; Kubota, Kengo

doi:10.1111/1462-2920.12745

Cited by 75 publications

(68 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The fact that amplification polymers carry up to hundreds of fluorophores (Choi et al, 2014) makes it possible to achieve high signal-to-background even when autofluorescence is high [e.g. in whole-mount vertebrate embryos (Choi et al, 2014;McLennan et al, 2015;Huss et al, 2015), in thick mouse brain sections (Sylwestrak et al, 2016) or in bacteria contained within environmental samples or other organisms (Rosenthal et al, 2013;Yamaguchi et al, 2015;Nikolakakis et al, 2015)]. The resulting HCR signal is stable for at least 1 week in zebrafish embryos stored in solution (Fig.…”

Section: Resultsmentioning

confidence: 99%

Mapping a multiplexed zoo of mRNA expression

et al. 2016

View full text Add to dashboard Cite

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffinembedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.

show abstract

Section: Resultsmentioning

confidence: 99%

Mapping a multiplexed zoo of mRNA expression

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Recently developed in situ DNA-HCR was also performed to enhance the signal detection [23]. However, reliable signals were still not obtained.…”

Section: Resultsmentioning

confidence: 99%

Phylogenetic diversity and in situ detection of eukaryotes in anaerobic sludge digesters

Matsubayashi

Shimada

et al. 2017

PLoS ONE

Self Cite

View full text Add to dashboard Cite

Eukaryotic communities in aerobic wastewater treatment processes are well characterized, but little is known about them in anaerobic processes. In this study, abundance, diversity and morphology of eukaryotes in anaerobic sludge digesters were investigated by quantitative real-time PCR (qPCR), 18S rRNA gene clone library construction and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Samples were taken from four different anaerobic sludge digesters in Japan. Results of qPCR of rRNA genes revealed that Eukarya accounted from 0.1% to 1.4% of the total number of microbial rRNA gene copy numbers. The phylogenetic affiliations of a total of 251 clones were Fungi, Alveolata, Viridiplantae, Amoebozoa, Rhizaria, Stramenopiles and Metazoa. Eighty-five percent of the clones showed less than 97.0% sequence identity to described eukaryotes, indicating most of the eukaryotes in anaerobic sludge digesters are largely unknown. Clones belonging to the uncultured lineage LKM11 in Cryptomycota of Fungi were most abundant in anaerobic sludge, which accounted for 50% of the total clones. The most dominant OTU in each library belonged to either the LKM11 lineage or the uncultured lineage A31 in Alveolata. Principal coordinate analysis indicated that the eukaryotic and prokaryotic community structures were related. The detection of anaerobic eukaryotes, including the members of the LKM11 and A31 lineages in anaerobic sludge digesters, by CARD-FISH revealed their sizes in the range of 2–8 μm. The diverse and uncultured eukaryotes in the LKM11 and the A31 lineages are common and ecologically relevant members in anaerobic sludge digester.

show abstract

“…The technique has potential for highly efficient amplification of short sequence oligonucleotides. For example, detection of Bacteria, Archaea and Methanosaetaceae in an anaerobic sludge sample was achieved by simultaneous in-situ DNA-HCR [135]. Analogously, an improved in-situ DNA HCR named quick HCR-FISH was tested for the rapid and sensitive identification of marine bacteria with low rRNA contents not only in seawater but also in sediment samples [136].…”

Section: Enhancing Performancementioning

confidence: 99%

Molecular Techniques for the Detection of Organisms in Aquatic Environments, with Emphasis on Harmful Algal Bloom Species

Medlin

Orozco

2017

Sensors

View full text Add to dashboard Cite

Molecular techniques to detect organisms in aquatic ecosystems are being gradually considered as an attractive alternative to standard laboratory methods. They offer faster and more accurate means of detecting and monitoring species, with respect to their traditional homologues based on culture and microscopic counting. Molecular techniques are particularly attractive when multiple species need to be detected and/or are in very low abundance. This paper reviews molecular techniques based on whole cells, such as microscope-based enumeration and Fluorescence In-Situ Hybridization (FISH) and molecular cell-free formats, such as sandwich hybridization assay (SHA), biosensors, microarrays, quantitative polymerase chain reaction (qPCR) and real time PCR (RT-PCR). Those that combine one or several laboratory functions into a single integrated system (lab-on-a-chip) and techniques that generate a much higher throughput data, such as next-generation systems (NGS), were also reviewed. We also included some other approaches that enhance the performance of molecular techniques. For instance, nano-bioengineered probes and platforms, pre-concentration and magnetic separation systems, and solid-phase hybridization offer highly pre-concentration capabilities. Isothermal amplification and hybridization chain reaction (HCR) improve hybridization and amplification techniques. Finally, we presented a study case of field remote sensing of harmful algal blooms (HABs), the only example of real time monitoring, and close the discussion with future directions and concluding remarks.

show abstract

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

Cited by 75 publications

References 28 publications

Mapping a multiplexed zoo of mRNA expression

Mapping a multiplexed zoo of mRNA expression

Phylogenetic diversity and in situ detection of eukaryotes in anaerobic sludge digesters

Molecular Techniques for the Detection of Organisms in Aquatic Environments, with Emphasis on Harmful Algal Bloom Species

Contact Info

Product

Resources

About