scholarly journals Quantitative Determination of Free-DNA Uptake in River Bacteria at the Single-Cell Level by In Situ Rolling-Circle Amplification

2006 ◽  
Vol 72 (9) ◽  
pp. 6248-6256 ◽  
Author(s):  
Fumito Maruyama ◽  
Katsuji Tani ◽  
Takehiko Kenzaka ◽  
Nobuyasu Yamaguchi ◽  
Masao Nasu
Keyword(s):  
Single Cell ◽  
Gene Amplification ◽  
Rolling Circle Amplification ◽  
Extracellular Dna ◽  
Single Cell Level ◽  
Dna Uptake ◽  
Cell Level ◽  
Indigenous Bacteria ◽  
Rolling Circle

ABSTRACT Detection of plasmid DNA uptake in river bacteria at the single-cell level was carried out by rolling-circle amplification (RCA). Uptake of a plasmid containing the green fluorescent protein gene (gfp) by indigenous bacteria from two rivers in Osaka, Japan, was monitored for 506 h using this in situ gene amplification technique with optimized cell permeabilization conditions. Plasmid uptake determined by in situ RCA was compared to direct counts of cells expressing gfp under fluorescence microscopy to examine differences in detection sensitivities between the two methods. Detection of DNA uptake as monitored by in situ RCA was 20 times higher at maximum than that by direct counting of gfp-expressing cells. In situ RCA could detect bacteria taking up the plasmid in several samples in which no gfp-expressing cells were apparent, indicating that in situ gene amplification techniques can be used to determine accurate rates of extracellular DNA uptake by indigenous bacteria in aquatic environments.

A DNAzyme-mediated target-initiated rolling circle amplification strategy based on microchip platform for the detection of apurinic/apyrimidinic endonuclease 1 at single-cell level

2021 ◽  
Author(s):  
Chunhuan Xu ◽  
Shengyu Chen ◽  
Jingjin Zhao ◽  
Xiaoshu Luo ◽  
Shulin Zhao
Keyword(s):  
Single Cell ◽  
Signal Amplification ◽  
Rolling Circle Amplification ◽  
Single Cell Level ◽  
Cell Level ◽  
Rolling Circle ◽  
Amplification Strategy

A DNAzyme-mediated target-initiated rolling circle signal amplification strategy based on microchip platform was developed for detecting apurinic/apyrimidine endonuclease 1 (APE1) at the single-cell level. This strategy was applied for assay...

Development of a Direct In Situ PCR Method for Detection of Specific Bacteria in Natural Environments

1998 ◽  
Vol 64 (4) ◽  
pp. 1536-1540 ◽  
Author(s):  
Katsuji Tani ◽  
Ken Kurokawa ◽  
Masao Nasu
Keyword(s):  
Alkaline Phosphatase ◽  
Single Cell ◽  
Natural Environments ◽  
Single Cell Level ◽  
Bacterial Cells ◽  
Cell Level ◽  
In Situ Pcr ◽  
Glass Slides ◽  
Pcr Products

ABSTRACT We applied HNPP (2-hydroxy-3-naphthoic acid-2′-phenylanilide phosphate) to direct in situ PCR for the routine detection of specific bacterial cells at the single-cell level. PCR was performed on glass slides with digoxigenin-labeled dUTP. The digoxigenin-labeled PCR products were detected with alkaline phosphatase-labeled antidigoxigenin antibody and HNPP which was combined with Fast Red TR. A bright red fluorescent signal was produced from conversion to HNP (dephosphorylated form) by alkaline phosphatase. We used the ECOL DNA primer set for amplification of ribosomal DNA of Escherichia coli to identify cells specifically at the single-cell level in a bacterial mixture. High-contrast images were obtained under an epifluorescence microscope with in situ PCR. By image analysis,E. coli cells in polluted river water also were detected.

scholarly journals Transfer of a Phage T4 Gene into Enterobacteriaceae, Determined at the Single-Cell Level

2009 ◽  
Vol 76 (4) ◽  
pp. 1274-1277 ◽  
Author(s):  
Takehiko Kenzaka ◽  
Masao Nasu ◽  
Katsuji Tani
Keyword(s):  
Gene Transfer ◽  
Single Cell ◽  
Dna Amplification ◽  
Single Cell Level ◽  
Cell Level ◽  
Phage T4 ◽  
Gene Copies ◽  
Amplification Technique

ABSTRACT The transfer range of phage genes was investigated at the single-cell level by using an in situ DNA amplification technique. After absorption of phages, a phage T4 gene was maintained in the genomes of non-plaque-forming bacteria at frequencies of 10−2 gene copies per cell. The gene transfer decreased the mutation frequencies in nonhost recipients.

scholarly journals Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification

2005 ◽  
Vol 71 (12) ◽  
pp. 7933-7940 ◽  
Author(s):  
Fumito Maruyama ◽  
Takehiko Kenzaka ◽  
Nobuyasu Yamaguchi ◽  
Katsuji Tani ◽  
Masao Nasu
Keyword(s):  
Gene Sequence ◽  
Rolling Circle Amplification ◽  
Specific Gene ◽  
Toxin Gene ◽  
Dna Uptake ◽  
Fluorescent Intensity ◽  
Prolonged Incubation ◽  
Rolling Circle ◽  
E Coli

ABSTRACT Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 106-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.

scholarly journals Visualizing and isolating iron-reducing microorganisms at single cell level

2020 ◽  
Author(s):  
Cuifen Gan ◽  
Rongrong Wu ◽  
Yeshen Luo ◽  
Jianhua Song ◽  
Dizhou Luo ◽  
...  
Keyword(s):  
Single Cell ◽  
Iron Reduction ◽  
High Sensitivity ◽  
Ex Situ ◽  
Single Cell Level ◽  
Cell Level ◽  
Cell Sorter ◽  
Fluorescent Intensity

AbstractIron-reducing microorganisms (FeRM) play key roles in many natural and engineering processes. Visualizing and isolating FeRM from multispecies samples are essential to understand the in-situ location and geochemical role of FeRM. Here, we visualized FeRM by a “turn-on” Fe2+-specific fluorescent chemodosimeter (FSFC) with high sensitivity, selectivity and stability. This FSFC could selectively identify and locate active FeRM from either pure culture, co-culture of different bacteria or sediment-containing samples. Fluorescent intensity of the FSFC could be used as an indicator of Fe2+ concentration in bacterial cultures. By integrating FSFC with a single cell sorter, we obtained three FSFC-labeled cells from an enriched consortia and all of them were subsequently evidenced to be capable of iron-reduction and two unlabeled cells were evidenced to have no iron-reducing capability, further confirming the feasibility of the FSFC.ImportanceVisualization and isolation of FeRM from samples containing multispecies are commonly needed by researchers from different disciplines, such as environmental microbiology, environmental sciences and geochemistry. However, no available method has been reported. In this study, we provid a solution to visualize FeRM and evaluate their activity even at single cell level. Integrating with single cell sorter, FeRM can also be isolated from samples containing multispecies. This method can be used as a powerful tool to uncover the in-situ or ex-situ role of FeRM and their interactions with ambient microbes or chemicals.

scholarly journals In situelectro-sequencing in three-dimensional tissues

2021 ◽  
Author(s):  
Qiang Li ◽  
Zuwan Lin ◽  
Ren Liu ◽  
Xin Tang ◽  
Jiahao Huang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Three Dimensional ◽  
Cell Types ◽  
Developmental Trajectory ◽  
Single Cell Level ◽  
Cell Level ◽  
Cell Gene Expression ◽  
Cell Gene

AbstractPairwise mapping of single-cell gene expression and electrophysiology in intact three-dimensional (3D) tissues is crucial for studying electrogenic organs (e.g., brain and heart)1–5. Here, we introducein situelectro-sequencing (electro-seq), combining soft bioelectronics within situRNA sequencing to stably map millisecond-timescale cellular electrophysiology and simultaneously profile a large number of genes at single-cell level across 3D tissues. We appliedin situelectro-seq to 3D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) patches, precisely registering the CM gene expression with electrophysiology at single-cell level, enabling multimodalin situanalysis. Such multimodal data integration substantially improved the dissection of cell types and the reconstruction of developmental trajectory from spatially heterogeneous tissues. Using machine learning (ML)-based cross-modal analysis,in situelectro-seq identified the gene-to-electrophysiology relationship over the time course of cardiac maturation. Further leveraging such a relationship to train a coupled autoencoder, we demonstrated the prediction of single-cell gene expression profile evolution using long-term electrical measurement from the same cardiac patch or 3D millimeter-scale cardiac organoids. As exemplified by cardiac tissue maturation,in situelectro-seq will be broadly applicable to create spatiotemporal multimodal maps and predictive models in electrogenic organs, allowing discovery of cell types and gene programs responsible for electrophysiological function and dysfunction.

scholarly journals Laser cleavable probes for in situ multiplexed glycan detection by single cell mass spectrometry

2019 ◽  
Vol 10 (47) ◽  
pp. 10958-10962 ◽  
Author(s):  
Jing Han ◽  
Xi Huang ◽  
Huihui Liu ◽  
Jiyun Wang ◽  
Caiqiao Xiong ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Drug Resistance ◽  
Single Cell ◽  
Cell Mass ◽  
Ionization Efficiency ◽  
Single Cell Level ◽  
Cell Level ◽  
Cell Mass Spectrometry ◽  
The Relationship

A single-cell MS approach for multiplexed glycan detection to investigate the relationship between drug resistance and glycans at a single-cell level and quantify multiple glycans, overcoming the limit of low ionization efficiency of glycans.

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