The role of Nucleic Acid Mimics (NAMs) on FISH-based techniques and applications for microbial detection

Nachér-Vázquez, Montserrat; Santos, Bruno F.R.; Azevedo, Nuno F.; Cerqueira, Laura

doi:10.1016/j.micres.2022.127086

Cited by 9 publications

(10 citation statements)

References 174 publications

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“… b The use of probe delivery techniques, can overcome the need for fixation steps [ 195 ]. c Due to their neutrally charged and synthetic nature, the use of DNA mimics, namely peptide nucleic acid (PNA) may enhance probes diffusion, by the increased resistance to proteases and nucleases and more affinity to the target sequence [ 178 ]. …”

Section: Technologies For Biofilms Transcriptomic Analysesmentioning

confidence: 99%

“…FISH is a technique typically based on the complementary targeting of ribosomal RNA (rRNA) by fluorescently labeled oligonucleotide probes [ 177 , 178 ]. For the efficient access of the probe to the target sequence, the cells must be fixed/permeabilized, yet maintaining the cell integrity [ 178 , 179 ]. After the hybridization, the probe will label the target sequence under stringent conditions, exciting the fluorochrome.…”

Section: Technologies For Biofilms Transcriptomic Analysesmentioning

confidence: 99%

See 1 more Smart Citation

Ecology of Legionella pneumophila biofilms: The link between transcriptional activity and the biphasic cycle

Barbosa,

Azevedo,

Goeres

et al. 2024

Biofilm

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Section: Technologies For Biofilms Transcriptomic Analysesmentioning

confidence: 99%

Section: Technologies For Biofilms Transcriptomic Analysesmentioning

confidence: 99%

Ecology of Legionella pneumophila biofilms: The link between transcriptional activity and the biphasic cycle

Barbosa,

Azevedo,

Goeres

et al. 2024

Biofilm

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“…Typically, sequences of 16S/23S and 18S/28S rRNA are the preferential target for members of the Bacteria/Archaea and Eukarya domains, respectively, as these are universal and highly abundant. They are also composed of highly conserved regions as well as variable ones, which allows the design of probes with different cell specificity ( Stewart and Franklin, 2008 ; Patwardhan et al., 2014 ; Nácher-Vázquez et al., 2022 ).…”

Section: Fish To Spatially Locate Microorganisms In Biofilmsmentioning

confidence: 99%

“…The next step is the sample washing to remove all loosely bound or unbound labelled probes, conferring a higher detection specificity. Lastly, but still of huge importance, the result of hybridization is visualized by epifluorescence microscopy or CLSM ( Cerqueira et al., 2008 ; Almeida and Azevedo, 2021 ; Nácher-Vázquez et al., 2022 ).…”

Section: Fish To Spatially Locate Microorganisms In Biofilmsmentioning

confidence: 99%

Imaging biofilms using fluorescence in situ hybridization: seeing is believing

Barbosa

Miranda

Azevedo

et al. 2023

Front. Cell. Infect. Microbiol.

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Biofilms are complex structures with an intricate relationship between the resident microorganisms, the extracellular matrix, and the surrounding environment. Interest in biofilms is growing exponentially given its ubiquity in so diverse fields such as healthcare, environmental and industry. Molecular techniques (e.g., next-generation sequencing, RNA-seq) have been used to study biofilm properties. However, these techniques disrupt the spatial structure of biofilms; therefore, they do not allow to observe the location/position of biofilm components (e.g., cells, genes, metabolites), which is particularly relevant to explore and study the interactions and functions of microorganisms. Fluorescence in situ hybridization (FISH) has been arguably the most widely used method for an in situ analysis of spatial distribution of biofilms. In this review, an overview on different FISH variants already applied on biofilm studies (e.g., CLASI-FISH, BONCAT-FISH, HiPR-FISH, seq-FISH) will be explored. In combination with confocal laser scanning microscopy, these variants emerged as a powerful approach to visualize, quantify and locate microorganisms, genes, and metabolites inside biofilms. Finally, we discuss new possible research directions for the development of robust and accurate FISH-based approaches that will allow to dig deeper into the biofilm structure and function.

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“…The FISH method robustness and implementation as molecular diagnostic tool greatly improves when using NAM's, which have enhanced sensitivity and specificity, when compared to DNA or RNA probes (Nacher-Vazquez et al, 2022). Moreover, when combined with microfluidics, the analysis time is reduced when compared to standard FISH method (Chien-Hsuan et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Microfluidics combined with fluorescence in situ hybridization (FISH) for Candida spp. detection

Barbosa

Rodrigues

Cerqueira

et al. 2022

Front. Bioeng. Biotechnol.

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One of the most prevalent healthcare-associated infection is the urinary tract infection (UTI), caused by opportunistic pathogens such as Candida albicans or non-albicans Candida species (NACS). Urine culture methods are routinely used for UTI diagnostics due to their specificity, sensitivity and low-cost. However, these methods are also laborious, time- and reagent-consuming. Therefore, diagnostic methods relying on nucleic acids have been suggested as alternatives. Nucleic acid-based methods can provide results within 24 h and can be adapted to point-of-care (POC) detection. Here, we propose to combine fluorescence in situ hybridization (FISH) with a microfluidic platform for the detection of Candida spp. As a case study we used C. tropicalis, which is reported as the second most common NACS urine isolate obtained from patients suspected with UTI. The microfluidic platform proposed in this study relies on hydrodynamic trapping, and uses physical barriers (e.g., microposts) for the separation of target cells from the suspension. Using a specific peptide nucleic acid (PNA) probe, the FISH procedure was applied onto previously trapped C. tropicalis cells present inside the microfluidic platform. Fluorescence signal intensity of hybridized cells was captured directly under the epifluorescence microscope. Overall, the PNA probe successfully detected C. tropicalis in pure culture and artificial urine (AU) using FISH combined with the microfluidic platform. Our findings reveal that FISH using nucleic acid mimics (PNA) in combination with microfluidics is a reliable method for the detection of microorganisms such as C. tropicalis. As such, this work provides the basis for the development of a POC detection platform in the future.

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The role of Nucleic Acid Mimics (NAMs) on FISH-based techniques and applications for microbial detection

Cited by 9 publications

References 174 publications

Ecology of Legionella pneumophila biofilms: The link between transcriptional activity and the biphasic cycle

Ecology of Legionella pneumophila biofilms: The link between transcriptional activity and the biphasic cycle

Imaging biofilms using fluorescence in situ hybridization: seeing is believing

Microfluidics combined with fluorescence in situ hybridization (FISH) for Candida spp. detection

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