Spectral imaging and nucleic acid mimics fluorescence in situ hybridization (SI-NAM-FISH) for multiplex detection of clinical pathogens
The application of nucleic acid mimics (NAMs), such as locked nucleic acid (LNA) and 2′-O-methyl-RNA (2’OMe), has improved the performance of fluorescence in situ hybridization (FISH) methods for the detection/location of clinical pathogens since they provide design versatility and thermodynamic con...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-09-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.976639/full |
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| author | Andreia S. Azevedo Andreia S. Azevedo Andreia S. Azevedo Andreia S. Azevedo Ricardo M. Fernandes Ricardo M. Fernandes Ricardo M. Fernandes Ana R. Faria Oscar F. Silvestre Jana B. Nieder Chenguang Lou Jesper Wengel Carina Almeida Carina Almeida Carina Almeida Nuno F. Azevedo Nuno F. Azevedo |
| author_facet | Andreia S. Azevedo Andreia S. Azevedo Andreia S. Azevedo Andreia S. Azevedo Ricardo M. Fernandes Ricardo M. Fernandes Ricardo M. Fernandes Ana R. Faria Oscar F. Silvestre Jana B. Nieder Chenguang Lou Jesper Wengel Carina Almeida Carina Almeida Carina Almeida Nuno F. Azevedo Nuno F. Azevedo |
| author_sort | Andreia S. Azevedo |
| collection | DOAJ |
| description | The application of nucleic acid mimics (NAMs), such as locked nucleic acid (LNA) and 2′-O-methyl-RNA (2’OMe), has improved the performance of fluorescence in situ hybridization (FISH) methods for the detection/location of clinical pathogens since they provide design versatility and thermodynamic control. However, an important limitation of FISH techniques is the low number of distinguishable targets. The use of filters in fluorescence image acquisition limits the number of fluorochromes that can be simultaneously differentiated. Recent advances in fluorescence spectral image acquisition have allowed the unambiguous identification of several microorganisms in a single sample. In this work, we aimed to combine NAM-FISH and spectral image analysis to develop and validate a new FISH variant, the spectral imaging-NAM-FISH (SI-NAM-FISH), that allows a multiplexed, robust and rapid detection of clinical pathogens. In the first stage, to implement/validate the method, we have selected seven fluorochromes with distinct spectral properties and seven bacterial species (Pseudomonas aeruginosa, Citrobacter freundii, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter calcoaceticus). As a strong variation in fluorescence intensities is found between species and between fluorochromes, seven versions of a EUB LNA/2’OMe probe, each conjugated to one of seven fluorochromes, were used to rank species/fluorochromes by FISH and then optimize species/fluorochrome pairing. Then, final validation tests were performed using mixed populations to evaluate the potential of the technique for separating/quantifying the different targets. Overall, validation tests with different proportions of bacteria labeled with the respective fluorochrome have shown the ability of the method to correctly distinguish the species. |
| first_indexed | 2024-12-10T05:16:20Z |
| format | Article |
| id | doaj.art-827cea36aa284bb8a0f4066db87a9502 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-10T05:16:20Z |
| publishDate | 2022-09-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Microbiology |
| spelling | doaj.art-827cea36aa284bb8a0f4066db87a95022022-12-22T02:00:57ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-09-011310.3389/fmicb.2022.976639976639Spectral imaging and nucleic acid mimics fluorescence in situ hybridization (SI-NAM-FISH) for multiplex detection of clinical pathogensAndreia S. Azevedo0Andreia S. Azevedo1Andreia S. Azevedo2Andreia S. Azevedo3Ricardo M. Fernandes4Ricardo M. Fernandes5Ricardo M. Fernandes6Ana R. Faria7Oscar F. Silvestre8Jana B. Nieder9Chenguang Lou10Jesper Wengel11Carina Almeida12Carina Almeida13Carina Almeida14Nuno F. Azevedo15Nuno F. Azevedo16LEPABE - Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Porto, PortugalALiCE - Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugali3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, PortugalIPATIMUP-Instituto de Patologia e Imunologia Molecular, Universidade do Porto, Porto, PortugalLEPABE - Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Porto, PortugalALiCE - Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Porto, PortugalINIAV, IP-National Institute for Agrarian and Veterinary Research, Vila Do Conde, PortugalINL International Iberian Nanotechnology Laboratory, Av Mestre José Veiga s/n, Braga, PortugalINL International Iberian Nanotechnology Laboratory, Av Mestre José Veiga s/n, Braga, PortugalINL International Iberian Nanotechnology Laboratory, Av Mestre José Veiga s/n, Braga, PortugalBiomolecular Nanoscale Engineering Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, Odense, DenmarkBiomolecular Nanoscale Engineering Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, Odense, DenmarkLEPABE - Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Porto, PortugalALiCE - Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Porto, PortugalINIAV, IP-National Institute for Agrarian and Veterinary Research, Vila Do Conde, PortugalLEPABE - Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Porto, PortugalALiCE - Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Porto, PortugalThe application of nucleic acid mimics (NAMs), such as locked nucleic acid (LNA) and 2′-O-methyl-RNA (2’OMe), has improved the performance of fluorescence in situ hybridization (FISH) methods for the detection/location of clinical pathogens since they provide design versatility and thermodynamic control. However, an important limitation of FISH techniques is the low number of distinguishable targets. The use of filters in fluorescence image acquisition limits the number of fluorochromes that can be simultaneously differentiated. Recent advances in fluorescence spectral image acquisition have allowed the unambiguous identification of several microorganisms in a single sample. In this work, we aimed to combine NAM-FISH and spectral image analysis to develop and validate a new FISH variant, the spectral imaging-NAM-FISH (SI-NAM-FISH), that allows a multiplexed, robust and rapid detection of clinical pathogens. In the first stage, to implement/validate the method, we have selected seven fluorochromes with distinct spectral properties and seven bacterial species (Pseudomonas aeruginosa, Citrobacter freundii, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter calcoaceticus). As a strong variation in fluorescence intensities is found between species and between fluorochromes, seven versions of a EUB LNA/2’OMe probe, each conjugated to one of seven fluorochromes, were used to rank species/fluorochromes by FISH and then optimize species/fluorochrome pairing. Then, final validation tests were performed using mixed populations to evaluate the potential of the technique for separating/quantifying the different targets. Overall, validation tests with different proportions of bacteria labeled with the respective fluorochrome have shown the ability of the method to correctly distinguish the species.https://www.frontiersin.org/articles/10.3389/fmicb.2022.976639/fullnucleic acid mimicsfluorescence in situ hybridizationclinical pathogensbacteriamultiplex detectionepifluorescence microscope |
| spellingShingle | Andreia S. Azevedo Andreia S. Azevedo Andreia S. Azevedo Andreia S. Azevedo Ricardo M. Fernandes Ricardo M. Fernandes Ricardo M. Fernandes Ana R. Faria Oscar F. Silvestre Jana B. Nieder Chenguang Lou Jesper Wengel Carina Almeida Carina Almeida Carina Almeida Nuno F. Azevedo Nuno F. Azevedo Spectral imaging and nucleic acid mimics fluorescence in situ hybridization (SI-NAM-FISH) for multiplex detection of clinical pathogens Frontiers in Microbiology nucleic acid mimics fluorescence in situ hybridization clinical pathogens bacteria multiplex detection epifluorescence microscope |
| title | Spectral imaging and nucleic acid mimics fluorescence in situ hybridization (SI-NAM-FISH) for multiplex detection of clinical pathogens |
| title_full | Spectral imaging and nucleic acid mimics fluorescence in situ hybridization (SI-NAM-FISH) for multiplex detection of clinical pathogens |
| title_fullStr | Spectral imaging and nucleic acid mimics fluorescence in situ hybridization (SI-NAM-FISH) for multiplex detection of clinical pathogens |
| title_full_unstemmed | Spectral imaging and nucleic acid mimics fluorescence in situ hybridization (SI-NAM-FISH) for multiplex detection of clinical pathogens |
| title_short | Spectral imaging and nucleic acid mimics fluorescence in situ hybridization (SI-NAM-FISH) for multiplex detection of clinical pathogens |
| title_sort | spectral imaging and nucleic acid mimics fluorescence in situ hybridization si nam fish for multiplex detection of clinical pathogens |
| topic | nucleic acid mimics fluorescence in situ hybridization clinical pathogens bacteria multiplex detection epifluorescence microscope |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.976639/full |
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