Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples

Detalhes bibliográficos
Autor(a) principal: Almeida, Carina
Data de Publicação: 2010
Outros Autores: Azevedo, N. F., Fernandes, R. M., Keevil, C. W., Vieira, M. J.
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/1822/16654
Resumo: A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. Specificity and sensitivity probe matching theoretical estimates were both 100%. The PNA FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species, confirmed the predicted theoretical values of specificity and sensitivity. The PNA FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. By performing a previous enrichment step, the PNA FISH method was able to detect 1 CFU per 10 mL of blood (5x109 ± 5x108 CFU/ml after an overnight enrichment step) and also 1 CFU per 10g of PIF (2x107 ± 5x106 CFU/ml after an 8h enrichment step),. The feces and water samples were also enriched according to the corresponding ISO methods, and results showed that the PNA FISH method was able to detect Salmonella immediately after conducting the first enrichment step. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4',6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples, taking less than 20 hours to obtain a diagnosis, except for PIF samples where the analysis takes less than 12 hours. This procedure may be used for food processing and municipal waters control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity.
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spelling Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samplesScience & TechnologyA fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. Specificity and sensitivity probe matching theoretical estimates were both 100%. The PNA FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species, confirmed the predicted theoretical values of specificity and sensitivity. The PNA FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. By performing a previous enrichment step, the PNA FISH method was able to detect 1 CFU per 10 mL of blood (5x109 ± 5x108 CFU/ml after an overnight enrichment step) and also 1 CFU per 10g of PIF (2x107 ± 5x106 CFU/ml after an 8h enrichment step),. The feces and water samples were also enriched according to the corresponding ISO methods, and results showed that the PNA FISH method was able to detect Salmonella immediately after conducting the first enrichment step. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4',6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples, taking less than 20 hours to obtain a diagnosis, except for PIF samples where the analysis takes less than 12 hours. This procedure may be used for food processing and municipal waters control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity.This work was supported by the Portuguese Institute Fundacao para a Ciencia e Tecnologia (Postdoctoral Fellowship SFRH/BPD/20484/2004 and Ph.D. Fellowship SFRH/BD/29297/2006).American Society for Microbiology (ASM)Universidade do MinhoAlmeida, CarinaAzevedo, N. F.Fernandes, R. M.Keevil, C. W.Vieira, M. J.20102010-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/1822/16654eng0099-224010.1128/AEM.01678-0920453122http://www.asm.org/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:RCAAP2023-07-21T12:35:33Zoai:repositorium.sdum.uminho.pt:1822/16654Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:31:25.106153Repositório 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 Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples
title Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples
spellingShingle Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples
Almeida, Carina
Science & Technology
title_short Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples
title_full Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples
title_fullStr Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples
title_full_unstemmed Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples
title_sort Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples
author Almeida, Carina
author_facet Almeida, Carina
Azevedo, N. F.
Fernandes, R. M.
Keevil, C. W.
Vieira, M. J.
author_role author
author2 Azevedo, N. F.
Fernandes, R. M.
Keevil, C. W.
Vieira, M. J.
author2_role author
author
author
author
dc.contributor.none.fl_str_mv Universidade do Minho
dc.contributor.author.fl_str_mv Almeida, Carina
Azevedo, N. F.
Fernandes, R. M.
Keevil, C. W.
Vieira, M. J.
dc.subject.por.fl_str_mv Science & Technology
topic Science & Technology
description A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. Specificity and sensitivity probe matching theoretical estimates were both 100%. The PNA FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species, confirmed the predicted theoretical values of specificity and sensitivity. The PNA FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. By performing a previous enrichment step, the PNA FISH method was able to detect 1 CFU per 10 mL of blood (5x109 ± 5x108 CFU/ml after an overnight enrichment step) and also 1 CFU per 10g of PIF (2x107 ± 5x106 CFU/ml after an 8h enrichment step),. The feces and water samples were also enriched according to the corresponding ISO methods, and results showed that the PNA FISH method was able to detect Salmonella immediately after conducting the first enrichment step. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4',6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples, taking less than 20 hours to obtain a diagnosis, except for PIF samples where the analysis takes less than 12 hours. This procedure may be used for food processing and municipal waters control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity.
publishDate 2010
dc.date.none.fl_str_mv 2010
2010-01-01T00:00:00Z
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/1822/16654
url http://hdl.handle.net/1822/16654
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 0099-2240
10.1128/AEM.01678-09
20453122
http://www.asm.org/
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 American Society for Microbiology (ASM)
publisher.none.fl_str_mv American Society for Microbiology (ASM)
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
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instacron_str RCAAP
institution RCAAP
reponame_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
repository.mail.fl_str_mv
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