Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples
Autor(a) principal: | |
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Data de Publicação: | 2010 |
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/1822/16654 |
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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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 instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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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1799132822910271488 |