Photothermal Mediated Rolling Circle Amplification toward Specific and Direct In Situ mRNA Detection

2021 ◽  
pp. 113507
Author(s):  
Dongdong Liu ◽  
Wenhua Li ◽  
Mingzhu Yang ◽  
Lizhen Qiu ◽  
Hongru Pian ◽  
...  
2014 ◽  
Vol 126 (9) ◽  
pp. 2421-2425 ◽  
Author(s):  
Ruijie Deng ◽  
Longhua Tang ◽  
Qianqian Tian ◽  
Ying Wang ◽  
Lei Lin ◽  
...  

2005 ◽  
Vol 71 (12) ◽  
pp. 7933-7940 ◽  
Author(s):  
Fumito Maruyama ◽  
Takehiko Kenzaka ◽  
Nobuyasu Yamaguchi ◽  
Katsuji Tani ◽  
Masao Nasu

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.


2018 ◽  
Vol 1039 ◽  
pp. 108-115 ◽  
Author(s):  
Yimei Feng ◽  
Yuna Guo ◽  
Yiran Li ◽  
Jing Tao ◽  
Lin Ding ◽  
...  

2001 ◽  
Vol 70 (3) ◽  
pp. 281-288 ◽  
Author(s):  
Yi Zhou ◽  
Margaret Calciano ◽  
Stefan Hamann ◽  
J.H. Leamon ◽  
Tod Strugnell ◽  
...  

2019 ◽  
Author(s):  
Hirokazu Takahashi ◽  
Kyohei Horio ◽  
Setsu Kato ◽  
Toshiro Kobori ◽  
Kenshi Watanabe ◽  
...  

ABSTRACTMeta-analyses using next generation sequencing is a powerful strategy for studying microbiota; however, it cannot clarify the role of individual microbes within microbiota. To know which cell expresses what gene is important for elucidation of the individual cell’s function in microbiota. In this report, we developed novel fluorescence in situ hybridization (FISH) procedure using RNase-H-assisted rolling circle amplification to visualize mRNA of interest in microbial cells without reverse transcription. Our results show that this method is applicable to both gram-negative and gram-positive microbes without any noise from DNA, and it is possible to visualize the target mRNA expression directly at the single-cell level. Therefore, our procedure, when combined with data of meta-analyses, can help to understand the role of individual microbes in the microbiota.


2006 ◽  
Vol 72 (9) ◽  
pp. 6248-6256 ◽  
Author(s):  
Fumito Maruyama ◽  
Katsuji Tani ◽  
Takehiko Kenzaka ◽  
Nobuyasu Yamaguchi ◽  
Masao Nasu

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.


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