Detection of Papillomavirus Nucleic Acids in Genital Precancers with the In Situ Hybridization Technique

We have been investigating the spatial distribution of nucleic acids intracellularly using in situ hybridization. The use of non-isotopic nucleotide analogs incorporated into the DNA probe allows the detection of the probe at its site of hybridization within the cell. This approach therefore is compatible with the high resolution available by electron microscopy. Biotinated or digoxigenated probe can be detected by antibodies conjugated to colloidal gold. Because mRNA serves as a template for the probe fragments, the colloidal gold particles are detected as arrays which allow it to be unequivocally distinguished from background.

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In situ hybridization at light and electron microscopic levels: identification of human papillomavirus nucleic acids

Pathology ◽

10.3109/00313029209063631 ◽

1992 ◽

Vol 24 (2) ◽

pp. 91-98 ◽

Cited By ~ 12

Author(s):

Kankatsu Yun ◽

Mark J. Sherwood

Keyword(s):

In Situ Hybridization ◽

Human Papillomavirus ◽

Nucleic Acids ◽

Electron Microscopic

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Combination of Direct Viable Count and Fluorescent In Situ Hybridization (DVC-FISH) as a Potential Method for Identifying Viable Vibrio parahaemolyticus in Oysters and Mussels

Foods ◽

10.3390/foods10071502 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1502

Author(s):

Jorge García-Hernández ◽

Manuel Hernández ◽

Yolanda Moreno

Keyword(s):

In Situ Hybridization ◽

Vibrio Parahaemolyticus ◽

Fluorescent In Situ Hybridization ◽

Potential Method ◽

Viable Count ◽

Hybridization Technique ◽

Fish Technique ◽

Viable Cells

Vibrio parahaemolyticus is a human food-borne pathogen with the ability to enter the food chain. It is able to acquire a viable, non-cultivable state (VBNC), which is not detected by traditional methods. The combination of the direct viable count method and a fluorescent in situ hybridization technique (DVC-FISH) makes it possible to detect microorganisms that can present VBNC forms in complex samples The optimization of the in vitro DVC-FISH technique for V. parahaemolyticus was carried out. The selected antibiotic was ciprofloxacin at a concentration of 0.75 μg/mL with an incubation time in DVC broth of 5 h. The DVC-FISH technique and the traditional plate culture were applied to detect and quantify the viable cells of the affected pathogen in artificially contaminated food matrices at different temperatures. The results obtained showed that low temperatures produced an important logarithmic decrease of V. parahaemolyticus, while at 22 °C, it proliferated rapidly. The DVC-FISH technique proved to be a useful tool for the detection and quantification of V. parahaemolyticus in the two seafood matrices of oysters and mussels. This is the first study in which this technique has been developed to detect viable cells for this microorganism.

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Microbial ecology of sulfate-reducing bacteria in wastewater biofilms analyzed by microelectrodes and fish (fluorescent in situ hybridization) technique

Water Science & Technology ◽

10.2166/wst.1999.0323 ◽

1999 ◽

Vol 39 (7) ◽

pp. 41-47 ◽

Cited By ~ 3

Author(s):

Satoshi Okabe ◽

Hisashi Satoh ◽

Tsukasa Itoh ◽

Yoshimasa Watanabe

Keyword(s):

In Situ Hybridization ◽

Sulfate Reduction ◽

Sulfate Reducing Bacteria ◽

Hybridization Technique ◽

Concentration Profiles ◽

Reduction Zone ◽

Sulfate Reducing ◽

Reducing Bacteria ◽

Culture Dependent

The vertical distribution of sulfate-reducing bacteria (SRB) in microaerophilic wastewater biofilms grown on fully submerged rotating disk reactors (RDR) was determined by the conventional culture-dependent MPN method and in situ hybridization of fluorescently-labelled 16S rRNA-targeted oligonucleotide probes for SRB in parallel. Chemical concentration profiles within the biofilm were also measured using microelectrodes for O2, S2-, NO3- and pH. In situ hybridization revealed that the SRB probe-stained cells were distributed throughout the biofilm even in the oxic surface zone in all states from single scattered cells to clustered cells. The higher fluorescence intensity and abundance of SRB probe-stained cells were found in the middle part of the biofilm. This result corresponded well with O2 and H2S concentration profiles measured by microelectrodes, showing sulfate reduction was restricted to a narrow anaerobic zone located about 500 μm below the biofilm surface. Results of the MPN and potential sulfate reducing activity (culture-dependent approaches) indicated a similar distribution of cultivable SRB in the biofilm. The majority of the general SRB probe-stained cells were hybridized with SRB 660 probe, suggesting that one important member of the SRB in the wastewater biofilm could be the genus Desulfobulbus. An addition of nitrate forced the sulfate reduction zone deeper in the biofilm and reduced the specific sulfate reduction rate as well. The sulfate reduction zone was consequently separated from O2 and NO3- respiration zones. Anaerobic H2S oxidation with NO3- was also induced by addition of nitrate to the medium.

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The usefulness of two-colour fluorescence in situ hybridization technique using chromosomal subset at metaphase and interphase stage in radiation biodosimetry

Indian Journal of Science and Technology ◽

10.17485/ijst/2009/v2i4.1 ◽

2009 ◽

Vol 2 (4) ◽

pp. 1-11

Author(s):

Kimio Tanaka

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Hybridization Technique ◽

Radiation Biodosimetry

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Fluorescence in-situ hybridization technique as a diagnostic and prognostic tool in oral squamous cell carcinoma

Journal of Oral and Maxillofacial Pathology ◽

10.4103/0973-029x.110731 ◽

2013 ◽

Vol 17 (1) ◽

pp. 61

Author(s):

PM Sunil ◽

CR Ramachandran ◽

S Gokul ◽

N Jaisanghar

Keyword(s):

Squamous Cell Carcinoma ◽

In Situ Hybridization ◽

Oral Squamous Cell Carcinoma ◽

Fluorescence In Situ Hybridization ◽

Cell Carcinoma ◽

Squamous Cell ◽

Hybridization Technique ◽

Prognostic Tool

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Physical mapping of barley genes using an ultrasensitive fluorescence in situ hybridization technique

Genome ◽

10.1139/g03-084 ◽

2004 ◽

Vol 47 (1) ◽

pp. 179-189 ◽

Cited By ~ 29

Author(s):

J L Stephens ◽

S E Brown ◽

N L.V Lapitan ◽

D L Knudson

Keyword(s):

In Situ Hybridization ◽

Hordeum Vulgare ◽

Fluorescence In Situ Hybridization ◽

Linkage Map ◽

Physical Mapping ◽

Physical Map ◽

Primary Objective ◽

Hybridization Technique ◽

Hordeum Vulgare L

The primary objective of this study was to elucidate gene organization and to integrate the genetic linkage map for barley (Hordeum vulgare L.) with a physical map using ultrasensitive fluorescence in situ hybridization (FISH) techniques for detecting signals from restriction fragment length polymorphism (RFLP) clones. In the process, a single landmark plasmid, p18S5Shor, was constructed that identified and oriented all seven of the chromosome pairs. Plasmid p18S5Shor was used in all hybridizations. Fourteen cDNA probes selected from the linkage map for barley H. vulgare 'Steptoe' × H. vulgare 'Morex' (Kleinhofs et al. 1993) were mapped using an indirect tyramide signal amplification technique and assigned to a physical location on one or more chromosomes. The haploid barley genome is large and a complete physical map of the genome is not yet available; however, it was possible to integrate the linkage map and the physical locations of these cDNAs. An estimate of the ratio of base pairs to centimorgans was an average of 1.5 Mb/cM in the distal portions of the chromosome arms and 89 Mb/cM near the centromere. Furthermore, while it appears that the current linkage maps are well covered with markers along the length of each arm, the physical map showed that there are large areas of the genome that have yet to be mapped.Key words: Hordeum vulgare, barley, physical mapping, FISH, cDNA, genetics, linkage, chromosome, BACs.

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Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization

Molecular and Cellular Biology ◽

10.1128/mcb.2.3.308-319.1982 ◽

1982 ◽

Vol 2 (3) ◽

pp. 308-319

Author(s):

G M Wahl ◽

L Vitto ◽

R A Padgett ◽

G R Stark

Keyword(s):

In Situ Hybridization ◽

Syrian Hamster ◽

Specific Radioactivity ◽

Large Chromosome ◽

Hybridization Technique ◽

Wild Type ◽

Aspartate Transcarbamylase ◽

Metaphase Chromosomes ◽

Cad Gene

Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional protein CAD, overproduce this protein as a result of amplification of the CAD gene. We have used a sensitive in situ hybridization technique to localize CAD genomes in spreads of metaphase chromosomes from several independent PALA-resistant lines and from wild-type PALA-sensitive cells. The amplified genes were always found within chromosomes, usually in an expanded region of the short arm of chromosome B9. In wild-type cells, the CAD gene was also on the short arm of chromosome B9. In one mutant line, 90 to 100 CAD genes were found within an expanded B9 chromosome and 10 to 15 more were near the distal end of one arm of several different chromosomes. Another line contained most the genes in a telomeric chromosome or large chromosome fragment. The amplified genes were in chromosomal regions that were stained in a banded pattern by trypsin-Giemsa. A few double minute chromosomes were observed in a very small fraction of the total spreads examined. The it situ hybridizations were performed in the presence of 10% dextral sulfate 500, which increases the signal by as much as 100-fold. Using recombinant DNA plasmids nick-translated with [125I]dCTP to high specific radioactivity, 10 CAD genes in a single chromosomal region were revealed after 1 week of autoradiographic exposure, and the position of the unique gene could be seen after 1 month.

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Allele-specific in situ hybridization (ASISH) analysis: a novel technique which resolves differential allelic usage of H19 within the same cell lineage during human placental development

Development ◽

10.1242/dev.122.3.839 ◽

1996 ◽

Vol 122 (3) ◽

pp. 839-847 ◽

Cited By ~ 1

Author(s):

G.I. Adam ◽

H. Cui ◽

S.J. Miller ◽

F. Flam ◽

R. Ohlsson

Keyword(s):

In Situ Hybridization ◽

First Trimester ◽

Paternal Allele ◽

Monoallelic Expression ◽

Hybridization Technique ◽

Placental Development ◽

Foetal Development ◽

Biallelic Expression ◽

Normal Human

Precursory studies of H19 transcription during human foetal development have demonstrated maternally derived monoallelic expression. Analyses in extra-embryonic tissues, however, have been more equivocal, with discernible levels of expression of the paternal allele of H19 documented in the first trimester placenta. By refining the in situ hybridization technique we have developed an assay to enable the functional imprinting status of H19 to be determined at the cellular level. This assay involves the use of oligonucleotide DNA probes that are able to discriminate between allelic RNA transcripts containing sequence polymorphisms. Biallelic expression of H19 is confined to a subpopulation of cells of the trophoblast lineage, the extravillous cytotrophoblast, while the mesenchymal stroma cells maintain the imprinted pattern of monoallelic expression of H19 throughout placental development. This data demonstrates that the low level of paternal H19 expression previously detected in normal human placenta is not due to a random loss of functional imprinting, but appears to result from a developmentally regulated cell type-specific activation of the paternal allele. In addition, biallelic expression of H19 does not seem to affect the functional imprinting of the insulin-like growth factor II gene, which is monoallelically expressed at relatively high levels in the extra-villous cytotrophoblasts. These results imply that the allelic usage of these two genes in normal human placental development may not be directly analogous to the situation previously documented in the mouse embryo.

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