Optimizing locked nucleic acid/2-O-methyl-RNA fluorescence in situ hybridization (LNA/2OMe-FISH) procedure for bacterial detection
Autor(a) principal: | |
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Data de Publicação: | 2019 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/1822/60589 |
Resumo: | Despite the successful application of LNA/2OMe-FISH procedures for bacteria detection, there is a lack of knowledge on the properties that affect hybridization. Such information is crucial for the rational design of protocols. Hence, this work aimed to evaluate the effect of three essential factors on the LNA/2OMe hybridization stephybridization temperature, NaCl concentration and type and concentration of denaturant (formamide, ethylene carbonate and urea). This optimization was performed for 3 Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Citrobacter freundii) and 2 Gram-positive bacteria (Enterococcus faecalis and Staphylococcus epidermidis), employing the response surface methodology and a Eubacteria probe. In general, it was observed that a high NaCl concentration is beneficial (from 2 M to 5 M), regardless of the denaturant used. Urea, formamide and ethylene carbonate are suitable denaturants for LNA/2OMe-FISH applications; but urea provides higher fluorescence intensities among the different bacteria, especially for gram-positive bacteria and for P. aeruginosa. However, a unique optimal protocol was not found for all tested bacteria. Despite this, the results indicate that a hybridization solution with 2 M of urea and 4 M of NaCl would be a proper starting point. Furthermore, a hybridization temperature around 62°C, for 14 bp probes with LNA monomers at every third position of 2?OMe and 64% of GC content, should be use in initial optimization of new LNA/2OMe-FISH protocols. |
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Optimizing locked nucleic acid/2-O-methyl-RNA fluorescence in situ hybridization (LNA/2OMe-FISH) procedure for bacterial detectionScience & TechnologyDespite the successful application of LNA/2OMe-FISH procedures for bacteria detection, there is a lack of knowledge on the properties that affect hybridization. Such information is crucial for the rational design of protocols. Hence, this work aimed to evaluate the effect of three essential factors on the LNA/2OMe hybridization stephybridization temperature, NaCl concentration and type and concentration of denaturant (formamide, ethylene carbonate and urea). This optimization was performed for 3 Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Citrobacter freundii) and 2 Gram-positive bacteria (Enterococcus faecalis and Staphylococcus epidermidis), employing the response surface methodology and a Eubacteria probe. In general, it was observed that a high NaCl concentration is beneficial (from 2 M to 5 M), regardless of the denaturant used. Urea, formamide and ethylene carbonate are suitable denaturants for LNA/2OMe-FISH applications; but urea provides higher fluorescence intensities among the different bacteria, especially for gram-positive bacteria and for P. aeruginosa. However, a unique optimal protocol was not found for all tested bacteria. Despite this, the results indicate that a hybridization solution with 2 M of urea and 4 M of NaCl would be a proper starting point. Furthermore, a hybridization temperature around 62°C, for 14 bp probes with LNA monomers at every third position of 2?OMe and 64% of GC content, should be use in initial optimization of new LNA/2OMe-FISH protocols.This work was financially supportedby project POCI-01-0145-FEDER-006939 (Laboratory for Process Engineering, Environment, Biotechnology and Energy – UID/EQU/00511/2013),POCI-01-0145-FEDER-006684 (Center of Biological Engineering - UID/BIO/04469) funded by EuropeanRegional Development Fund (ERDF) through COMPETE2020– Programa Operacional Competitividade e Internacionalização (POCI), and by national funds (PIDDAC) through FCT – Fundação para a Ciência e a Tecnologia/MCTES; NORTE-01-0145-FEDER-000004- BioTecNorte operation,funded by the EuropeanRegional Development Fund under the scope of Norte2020 Programa Operacional Regional do Norte; PostDoctoral Fellowship SFRH/BPD/121601/2016 (Andreia S. Azevedo) and PhD Fellowship SFRH/ BD/118860/2016 (Ricardo M. Fernandes) supportedby national funds throughFCT Fundação para a Ciência e a Tecnologia; Project PTDC/DTP-PIC/4562/2014 – POCI-01-0145FEDER-016678 (Coded-FISH) and Project POCI01-0145-FEDER-030431(CLASInVivo), funded by FEDER funds through COMPETE2020- Programa Operacional Competitividade e Internacionalização (POCI) and by national funds through FCT Fundação para a Ciência e a Tecnologia, I.P.info:eu-repo/semantics/publishedVersionPublic Library of ScienceUniversidade do MinhoAzevedo, Andreia S.Sousa, Inês M.Fernandes, R.Azevedo, Nuno F.Almeida, Carina2019-05-312019-05-31T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/1822/60589engAzevedo, Andreia S.; Sousa, Inês M.; Fernandes, R.; Azevedo, Nuno F.; Almeida, Carina, Optimizing locked nucleic acid/2-O-methyl-RNA fluorescence in situ hybridization (LNA/2OMe-FISH) procedure for bacterial detection. PLoS One, 14(5), e0217689, 20191932-62031932-620310.1371/journal.pone.021768931150460http://journals.plos.org/plosone/info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-07-21T12:29:49Zoai:repositorium.sdum.uminho.pt:1822/60589Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:24:52.095228Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Optimizing locked nucleic acid/2-O-methyl-RNA fluorescence in situ hybridization (LNA/2OMe-FISH) procedure for bacterial detection |
title |
Optimizing locked nucleic acid/2-O-methyl-RNA fluorescence in situ hybridization (LNA/2OMe-FISH) procedure for bacterial detection |
spellingShingle |
Optimizing locked nucleic acid/2-O-methyl-RNA fluorescence in situ hybridization (LNA/2OMe-FISH) procedure for bacterial detection Azevedo, Andreia S. Science & Technology |
title_short |
Optimizing locked nucleic acid/2-O-methyl-RNA fluorescence in situ hybridization (LNA/2OMe-FISH) procedure for bacterial detection |
title_full |
Optimizing locked nucleic acid/2-O-methyl-RNA fluorescence in situ hybridization (LNA/2OMe-FISH) procedure for bacterial detection |
title_fullStr |
Optimizing locked nucleic acid/2-O-methyl-RNA fluorescence in situ hybridization (LNA/2OMe-FISH) procedure for bacterial detection |
title_full_unstemmed |
Optimizing locked nucleic acid/2-O-methyl-RNA fluorescence in situ hybridization (LNA/2OMe-FISH) procedure for bacterial detection |
title_sort |
Optimizing locked nucleic acid/2-O-methyl-RNA fluorescence in situ hybridization (LNA/2OMe-FISH) procedure for bacterial detection |
author |
Azevedo, Andreia S. |
author_facet |
Azevedo, Andreia S. Sousa, Inês M. Fernandes, R. Azevedo, Nuno F. Almeida, Carina |
author_role |
author |
author2 |
Sousa, Inês M. Fernandes, R. Azevedo, Nuno F. Almeida, Carina |
author2_role |
author author author author |
dc.contributor.none.fl_str_mv |
Universidade do Minho |
dc.contributor.author.fl_str_mv |
Azevedo, Andreia S. Sousa, Inês M. Fernandes, R. Azevedo, Nuno F. Almeida, Carina |
dc.subject.por.fl_str_mv |
Science & Technology |
topic |
Science & Technology |
description |
Despite the successful application of LNA/2OMe-FISH procedures for bacteria detection, there is a lack of knowledge on the properties that affect hybridization. Such information is crucial for the rational design of protocols. Hence, this work aimed to evaluate the effect of three essential factors on the LNA/2OMe hybridization stephybridization temperature, NaCl concentration and type and concentration of denaturant (formamide, ethylene carbonate and urea). This optimization was performed for 3 Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Citrobacter freundii) and 2 Gram-positive bacteria (Enterococcus faecalis and Staphylococcus epidermidis), employing the response surface methodology and a Eubacteria probe. In general, it was observed that a high NaCl concentration is beneficial (from 2 M to 5 M), regardless of the denaturant used. Urea, formamide and ethylene carbonate are suitable denaturants for LNA/2OMe-FISH applications; but urea provides higher fluorescence intensities among the different bacteria, especially for gram-positive bacteria and for P. aeruginosa. However, a unique optimal protocol was not found for all tested bacteria. Despite this, the results indicate that a hybridization solution with 2 M of urea and 4 M of NaCl would be a proper starting point. Furthermore, a hybridization temperature around 62°C, for 14 bp probes with LNA monomers at every third position of 2?OMe and 64% of GC content, should be use in initial optimization of new LNA/2OMe-FISH protocols. |
publishDate |
2019 |
dc.date.none.fl_str_mv |
2019-05-31 2019-05-31T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/1822/60589 |
url |
http://hdl.handle.net/1822/60589 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Azevedo, Andreia S.; Sousa, Inês M.; Fernandes, R.; Azevedo, Nuno F.; Almeida, Carina, Optimizing locked nucleic acid/2-O-methyl-RNA fluorescence in situ hybridization (LNA/2OMe-FISH) procedure for bacterial detection. PLoS One, 14(5), e0217689, 2019 1932-6203 1932-6203 10.1371/journal.pone.0217689 31150460 http://journals.plos.org/plosone/ |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Public Library of Science |
publisher.none.fl_str_mv |
Public Library of Science |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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