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1.
Methods Mol Biol ; 2292: 35-48, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33651350

RESUMO

Cystoscopy is considered the standard approach to the diagnostic workup of urinary symptoms. It has high sensitivity and specificity for papillary tumors of the bladder but low sensitivity and specificity for flat lesions. It is also expensive and may cause discomfort and complications. Urine cytology, in contrast, has the advantage of being a noninvasive test with high specificity but suffers from low sensitivity in low-grade and early-stage tumors, possibly due to the low number of exfoliated cells in urine. Numerous new noninvasive tests have been proposed. Among these, fluorescence in situ hybridization (FISH) has been studied for long time and in 2005 UroVysion Bladder Cancer Kit (UroVysion Kit) (Abbott/Vysis) received FDA approval for initial diagnosis of bladder carcinoma in patients with hematuria and subsequent monitoring for tumor recurrence in patients previously diagnosed with bladder cancer. The UroVysion Kit is designed to detect aneuploidy for chromosomes 3, 7, 17, and loss of the 9p21 locus by FISH in urine specimens from symptomatic patients, those with hematuria suspected of having bladder cancer. Here, the approach for FISH assay by using UroVysion Bladder Cancer kit according to manufacturer's instructions is described.


Assuntos
Hematúria/urina , Hibridização in Situ Fluorescente/métodos , Neoplasias da Bexiga Urinária/urina , Aneuploidia , Cromossomos Humanos/genética , Hematúria/genética , Humanos , Neoplasias da Bexiga Urinária/genética
2.
Methods Mol Biol ; 2292: 121-131, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33651357

RESUMO

Bladder cancer has a very high frequency of recurrence and therefore requires close clinical surveillance throughout its life, with cystoscopies and serial cytological examinations. These tests are both invasive and expensive, with considerable interpersonal and inter-institutional variability. Moreover, cytological examination used for the diagnosis of low-grade tumors has a low sensitivity; thus, there is an increasing focus on the research for new, accurate, urinary markers. Herein, the biological basis, methodologies, and diagnostic performance of biomarkers are discussed.


Assuntos
Neoplasias da Bexiga Urinária/urina , Antígenos de Neoplasias/urina , Biomarcadores Tumorais/urina , Humanos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Proteínas Nucleares/urina , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Neoplasias da Bexiga Urinária/diagnóstico
3.
Methods Mol Biol ; 2246: 1-15, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576979

RESUMO

Fluorescence in situ hybridization (FISH) is a molecular biology technique that enables the localization, quantification, and identification of microorganisms in a sample. This technique has found applications in several areas, most notably the environmental, for quantification and diversity assessment of microorganisms and, the clinical, for the rapid diagnostics of infectious agents. The FISH method is based on the hybridization of a fluorescently labeled nucleic acid probe with a complementary sequence that is present inside the microbial cell, typically in the form of ribosomal RNA (rRNA). In fact, an hybridized cell is typically only detectable because a large number of multiple fluorescent particles (as many as the number of target sequences available) are present inside the cell. Here, we will review the major steps involved in a standard FISH protocol, namely, fixation/permeabilization, hybridization, washing, and visualization/detection. For each step, the major variables/parameters are identified and, subsequently, their impact on the overall hybridization performance is assessed in detail.


Assuntos
Hibridização in Situ Fluorescente/métodos , Microbiota/genética , Fluorescência , Sondas de Ácido Nucleico/genética , Sondas de Oligonucleotídeos/genética , RNA Bacteriano/genética , RNA Ribossômico/genética
4.
Methods Mol Biol ; 2246: 17-33, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576980

RESUMO

FISH has gained an irreplaceable place in microbiology because of its ability to detect and locate a microorganism, or a group of organisms, within complex samples. However, FISH role has evolved drastically in the last few decades and its value has been boosted by several advances in signal intensity, imaging acquisitions, automation, method robustness, and, thus, versatility. This has resulted in a range of FISH variants that gave researchers the ability to access a variety of other valuable information such as complex population composition, metabolic activity, gene detection/quantification, or subcellular location of genetic elements. In this chapter, we will review the more relevant FISH variants, their intended use, and how they address particular challenges of classical FISH.


Assuntos
Hibridização in Situ Fluorescente/métodos , Automação/métodos
5.
Methods Mol Biol ; 2246: 35-50, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576981

RESUMO

Fluorescence in situ hybridization (FISH) is a well-established technique that allows the detection of microorganisms in diverse types of samples (e.g., clinical, food, environmental samples, and biofilm communities). The FISH probe design is an essential step in this technique. For this, two strategies can be used, the manual form based on multiple sequence alignment to identify conserved regions and programs/software specifically developed for the selection of the sequence of the probe. Additionally, databases/software for the theoretical evaluation of the probes in terms of specificity, sensitivity, and thermodynamic parameters (melting temperature and Gibbs free energy change) are used. The purpose of this chapter is to describe the essential steps and guidelines for the design of FISH probes (e.g., DNA and Nucleic Acid Mimic (NAM) probes), and its theoretical evaluation through the application of diverse bioinformatic tools.


Assuntos
Biologia Computacional/métodos , Hibridização in Situ Fluorescente/métodos , Sequência de Bases , Biofilmes/crescimento & desenvolvimento , DNA/genética , Fluorescência , Ácidos Nucleicos/genética , Sondas de Oligonucleotídeos/genética , Alinhamento de Sequência
6.
Methods Mol Biol ; 2246: 51-67, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576982

RESUMO

Fluorescence in situ hybridization (FISH) enables the detection and enumeration of microorganisms in a diversity of samples. Short-length oligonucleotide DNA probes complementary to 16S or 23S rRNA sequences are generally used to target different phylogenetic levels. The protocol for the application of FISH to aggregated or suspended cells in mixed microbial communities is described in this chapter, with a special emphasis on environmental samples.


Assuntos
Hibridização in Situ Fluorescente/métodos , Microbiota/genética , Sondas de Oligonucleotídeos/genética , Filogenia , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética
7.
Methods Mol Biol ; 2246: 87-96, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576984

RESUMO

Oligonucleotides able to hybridize bacterial RNA via in situ hybridization may potentially act as new antimicrobials, replacing antibiotics, and as fast in vivo diagnostic probes, outperforming current clinical methodologies. Nonetheless, oligonucleotides are not able to efficiently permeate the multi-layered bacterial envelope to reach their target RNA in the cytosol. Cationic fusogenic liposomes are here suggested as vehicles to enable the internalization of oligonucleotides in bacteria. Here, we describe the formulation of DOTAP-DOPE liposomes, their complexation with small negatively charged oligonucleotides, and the evaluation of the intracellular delivery of the oligonucleotides in bacteria. This strategy uncovers the potential of performing FISH in vivo for real-time detection and treatment of infections.


Assuntos
Bactérias/metabolismo , Lipossomos/química , Oligonucleotídeos/metabolismo , Cátions/química , Citosol/metabolismo , Ácidos Graxos Monoinsaturados/química , Hibridização in Situ Fluorescente/métodos , Fosfatidiletanolaminas/química , Compostos de Amônio Quaternário/química , RNA Bacteriano/metabolismo
8.
Methods Mol Biol ; 2246: 69-86, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576983

RESUMO

Traditionally, RNA and DNA probes are used in fluorescence in situ hybridization (FISH) methods for microbial detection and characterization of communities' structure and diversity. However, the recent introduction of nucleic acid mimics (NAMs) has improved the robustness of the FISH methods in terms of sensitivity and specificity. Several NAMs have been used, of which the most relevant are peptide nucleic acid (PNA), locked nucleic acids (LNA), 2'-O-methyl RNA (2'OMe), and phosphorothioates (PS). In this chapter, we describe a protocol using PNA and LNA/2'OMe probes for microbial detection by FISH, pointing out the differences between them. These protocols are easily adapted to different microorganisms and different probe sequences.


Assuntos
Hibridização in Situ Fluorescente/métodos , Ácidos Nucleicos/genética , Microbiota/genética , Sondas de Ácido Nucleico/genética , Oligonucleotídeos/genética , Ácidos Nucleicos Peptídicos/genética , Sensibilidade e Especificidade
9.
Methods Mol Biol ; 2246: 97-109, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576985

RESUMO

Biofilms are often composed of different bacterial and fungal species/strains, which form complex structures based on social interactions with each other. Fluorescence in situ hybridization (FISH) can help us identify the different species/strains present within a biofilm , and when coupled with confocal scanning laser microscopy (CSLM), it enables the visualization of the three-dimensional (3D) structure of the biofilm and the spatial arrangement of each individual species/strain within it. In this chapter, we describe the protocol for characterizing multistrain or multispecies biofilm formation using NAM-FISH and CSLM.


Assuntos
Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Hibridização in Situ Fluorescente/métodos , Microscopia Confocal/métodos , Ácidos Nucleicos/genética , Fluorescência
10.
Methods Mol Biol ; 2246: 111-128, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576986

RESUMO

High-resolution, spatial characterization of microbial communities is critical for the accurate understanding of microbe-microbe and microbe-plant interactions in leaf surfaces (phyllosphere). However, leaves are specially challenging surfaces for imaging methods due to their high autofluorescence. In this chapter we describe the Leaf-FISH method. Leaf-FISH is a fluorescence in situ hybridization (FISH) method specially adapted to the requirements of plant tissues. Leaf-FISH uses a combination of leaf pretreatments coupled with spectral imaging confocal microscopy and image post-processing to visualize bacterial taxa on a structural-informed context recreated from the residual background autofluorescence of the tissues. Leaf-FISH is suitable for simultaneous identification of multiple bacterial taxa using multiple taxon-specific fluorescently labeled oligonucleotide probes (combinatorial labeling).


Assuntos
Bactérias/crescimento & desenvolvimento , Hibridização in Situ Fluorescente/métodos , Folhas de Planta/microbiologia , Plantas/microbiologia , Bactérias/genética , Microscopia Confocal/métodos , Sondas de Oligonucleotídeos/genética
11.
Methods Mol Biol ; 2246: 129-140, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576987

RESUMO

CARD-FISH technique allows us to increase microbial cell detection compared to traditional FISH assays. Specific nonfluorescent oligonucleotide probes targeting 16S rRNA genes are employed and are chemically activated by the binding of tyramide molecules, with the latter able to generate a cascade of fluorescence signals, improving sensitivity and reducing background noise. The technique has been successfully applied for the detection of microorganisms in different environmental matrices and under different growth conditions (including those where cells are characterized by low physiological activity and low ribosome content). This chapter presents a straightforward procedure to execute CARD-FISH analysis, from sample preparation and fixation, to microscopic visualization, along with relevant technical notes.


Assuntos
Hibridização in Situ Fluorescente/métodos , Bactérias/genética , Catálise , Fluorescência , Sondas de Oligonucleotídeos/genética , RNA Ribossômico 16S/genética
12.
Methods Mol Biol ; 2246: 141-155, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576988

RESUMO

In this chapter we describe the use of fluorescent quantum dots (QDs) as labels for microbial mRNA transcripts using fluorescence in situ hybridization (FISH). Unlike organic dyes, which are the standard labels in modern FISH methods, QDs provide fluorescence signals that are much brighter and resistant to photobleaching, with an expanded spectral range for multiplexing. We describe the preparation of QDs with compact sizes necessary for accurate labeling, their application for analyzing lacZ transcripts in Escherichia coli cells using FISH, and an assessment of signal stability. We further discuss differences between methods for mammalian cells and bacteria, for which individual nucleic acids cannot be discretely counted due to the small cell size and the optical diffraction limit.


Assuntos
Escherichia coli/metabolismo , Hibridização in Situ Fluorescente/métodos , Pontos Quânticos/metabolismo , Animais , Fluorescência , Corantes Fluorescentes/metabolismo , Mamíferos/metabolismo , Fotodegradação , RNA Mensageiro/metabolismo
13.
Methods Mol Biol ; 2246: 157-168, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576989

RESUMO

The possibility of visualizing bacteriophage-host interactions through fluorescence in situ hybridization (FISH)-derived methods is gaining relevance in the last few years. These methods allow the possibility of discriminating between phage-infected and noninfected cells and the assessment of the different infection stages at the single cell level. In opposition to bacterial cells, the detection of phages is more challenging due to the low number of nucleic acid copies. However, by using a conserved region of the phage genome that is highly expressed during transcription, a FISH signal targeting phage DNA copies and mRNA transcripts can be easily visible inside the bacterial host cells.In this book chapter, we will cover both the design of locked nucleic acid (LNA) probes for phages and a FISH method for the detection of phages inside bacterial cells.


Assuntos
Bactérias/virologia , Bacteriófagos/genética , Hibridização in Situ Fluorescente/métodos , DNA/genética , Interações entre Hospedeiro e Microrganismos/genética , Oligonucleotídeos/genética , RNA Mensageiro/genética
14.
Methods Mol Biol ; 2246: 169-205, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576990

RESUMO

Direct-geneFISH is a Fluorescence In Situ Hybridization (FISH) method that directly links gene presence, and thus potential metabolic capabilities, to cell identity. The method uses rRNA-targeting oligonucleotide probes to identify cells and dsDNA polynucleotide probes carrying multiple molecules of the same fluorochrome to detect genes. In addition, direct-geneFISH allows quantification of the cell fraction carrying the targeted gene and the number of target genes per cell. It can be applied to laboratory cultures, for example, enrichments and phage infections, and to environmental samples. This book chapter describes the main steps of the direct-geneFISH protocol: probe design and synthesis, the "core" direct-geneFISH protocol and lastly, microscopy and data analysis.


Assuntos
Hibridização in Situ Fluorescente/métodos , Microbiota/genética , Análise de Célula Única/métodos , Bacteriófagos/genética , DNA/genética , Microscopia/métodos , Sondas de Oligonucleotídeos/genética , RNA Ribossômico/genética
15.
Methods Mol Biol ; 2246: 207-224, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576991

RESUMO

Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) is an imaging method used to identify microorganisms in environmental samples based on their phylogeny. CARD-FISH can be combined with nano-scale secondary ion mass spectrometry (nanoSIMS) to directly link the cell identity to their activity, measured as the incorporation of stable isotopes into hybridized cells after stable isotope probing. In environmental microbiology, a combination of these methods has been used to determine the identity and growth of uncultured microorganisms, and to explore the factors controlling their activity. Additionally, FISH-nanoSIMS has been widely used to directly visualize microbial interactions in situ. Here, we describe a step-by-step protocol for a combination of CARD-FISH, laser marking, and nanoSIMS analysis on samples from aquatic environments.


Assuntos
Hibridização in Situ Fluorescente/métodos , Espectrometria de Massa de Íon Secundário/métodos , Isótopos de Carbono/metabolismo , Microbiologia Ambiental , Marcação por Isótopo/métodos , Microbiota/genética , Microbiota/fisiologia , Isótopos de Nitrogênio/metabolismo , Filogenia
16.
Methods Mol Biol ; 2246: 225-236, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576992

RESUMO

Microautoradiography (MAR) is a technique by which assimilated radioactive tracers incorporated into the biomass can be detected by a film emulsion. This allows for the testing of cellular preferences in electron donors and acceptors of individual cells in complex microbial assemblages, as well as the ability to take up substrates under diverse environmental exposures.Combination with staining techniques such as fluorescence in situ hybridization (FISH) can be used to identify the involved cells. Here, the practical aspects of a combined microautoradiography and fluorescence in situ hybridization (MAR-FISH) approach are described.


Assuntos
Autorradiografia/métodos , Hibridização in Situ Fluorescente/métodos , Biomassa , Elétrons , Microbiota/genética , Filogenia
17.
Methods Mol Biol ; 2246: 237-247, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576993

RESUMO

A method for measuring mRNA copies in intact bacterial cells by fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) is presented. Unlike conventional single-molecule FISH, where the presence of a transcript is determined by fluorescence intensity, fliFISH relies on On-Off duty cycles of photo-switching dyes to set a predetermined threshold for distinguishing true signals from background noise. The method provides a quantitative approach for detecting and counting true mRNA copies and rejecting false signals with high accuracy.


Assuntos
Bactérias/genética , Hibridização in Situ Fluorescente/métodos , RNA Mensageiro/genética , Fluorescência , Corantes Fluorescentes/metabolismo , Microscopia de Fluorescência/métodos
18.
Methods Mol Biol ; 2246: 249-261, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576994

RESUMO

Suitable molecular methods for a faster microbial identification in food and clinical samples have been explored and optimized during the last decades. However, most molecular methods still rely on time-consuming enrichment steps prior to detection, so that the microbial load can be increased and reach the detection limit of the techniques.In this chapter, we describe an integrated methodology that combines a microfluidic (lab-on-a-chip) platform, designed to concentrate cell suspensions and speed up the identification process in Saccharomyces cerevisiae , and a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) protocol optimized and adapted to microfluidics. Microfluidic devices with different geometries were designed, based on computational fluid dynamics simulations, and subsequently fabricated in polydimethylsiloxane by soft lithography. The microfluidic designs and PNA-FISH procedure described here are easily adaptable for the detection of other microorganisms of similar size.


Assuntos
Hibridização in Situ Fluorescente/métodos , Microfluídica/métodos , Dispositivos Lab-On-A-Chip , Ácidos Nucleicos Peptídicos/genética , Saccharomyces cerevisiae/genética
19.
Methods Mol Biol ; 2246: 263-277, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576995

RESUMO

Flow-Fluorescence in situ hybridization (Flow-FISH) enables multiparametric high-throughput detection of target nucleic acid sequences at the single cell-level, allowing an accurate quantification of different cell populations by using a combination of flow cytometry and fluorescent in situ hybridization (FISH). In this chapter, a flow-FISH protocol is described with labeled nucleic acid mimics (NAMs) (e.g. LNA/2'OMe and PNA) acting as the reporter molecules. This protocol allows for the specific detection of bacterial cells. Hence, this protocol can be carried out with minor adjustments, in order to simultaneously detect different species of bacteria in different types of clinical, food, or environmental samples.


Assuntos
Bactérias/genética , Hibridização in Situ Fluorescente/métodos , Sondas de Ácido Nucleico/genética , Ácidos Nucleicos/genética , Oligonucleotídeos/genética
20.
Methods Mol Biol ; 2246: 279-290, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576996

RESUMO

Foodborne diseases are a major global public health concern. The gold standard detection techniques, namely culture plating techniques, are nowadays considered inadequate for the modern food industry mainly due to the time requirements of this sector. As such, the adoption of faster detection methods to be routinely used in screening the protocols of foodborne pathogens is required. Fluorescence in situ Hybridization (FISH) methods have been described as a valid alternative to standard plating techniques and are compatible with the requirements of the food industry.Here, we give an overview of the methodological aspects to consider regarding sample preparation and sample analysis for pathogen detection in food matrices by FISH methodologies.


Assuntos
Microbiologia de Alimentos/métodos , Hibridização in Situ Fluorescente/métodos , Técnicas Biossensoriais/métodos , Contaminação de Alimentos/prevenção & controle , Indústria Alimentícia/métodos
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