Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum

Detalhes bibliográficos
Autor(a) principal: Rubio, Marcela da Silva [UNESP]
Data de Publicação: 2017
Outros Autores: Penha Filho, Rafael Antonio Casarin [UNESP], Almeida, Adriana Maria de [UNESP], Berchieri, Angelo [UNESP]
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da UNESP
Texto Completo: http://dx.doi.org/10.1080/03079457.2017.1339866
http://hdl.handle.net/11449/169936
Resumo: Currently there are 2659 Salmonella serovars. The host-specific biovars Salmonella Pullorum and Salmonella Gallinarum cause systemic infections in food-producing and wild birds. Fast diagnosis is crucial to control the dissemination in avian environments. The present work describes the development of a multiplex qPCR in real time using a low-cost DNA dye (SYBr Green) to identify and quantify these biovars. Primers were chosen based on genomic regions of difference (RoD) and optimized to control dimers. Primers pSGP detect both host-specific biovars but not other serovars and pSG and pSP differentiate biovars. Three amplicons showed different melting temperatures (Tm), allowing differentiation. The pSGP amplicon (97 bp) showed Tm of 78°C for both biovars. The pSG amplicon (273 bp) showed a Tm of 86.2°C for S. Gallinarum and pSP amplicon (260 bp) dissociated at 84.8°C for S. Pullorum identification. The multiplex qPCR in real time showed high sensitivity and was capable of quantifying 108–101 CFU of these biovars.
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spelling Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella PullorumbiovarFowl typhoididentificationmelting curvepullorum diseaseRODSYBr greenCurrently there are 2659 Salmonella serovars. The host-specific biovars Salmonella Pullorum and Salmonella Gallinarum cause systemic infections in food-producing and wild birds. Fast diagnosis is crucial to control the dissemination in avian environments. The present work describes the development of a multiplex qPCR in real time using a low-cost DNA dye (SYBr Green) to identify and quantify these biovars. Primers were chosen based on genomic regions of difference (RoD) and optimized to control dimers. Primers pSGP detect both host-specific biovars but not other serovars and pSG and pSP differentiate biovars. Three amplicons showed different melting temperatures (Tm), allowing differentiation. The pSGP amplicon (97 bp) showed Tm of 78°C for both biovars. The pSG amplicon (273 bp) showed a Tm of 86.2°C for S. Gallinarum and pSP amplicon (260 bp) dissociated at 84.8°C for S. Pullorum identification. The multiplex qPCR in real time showed high sensitivity and was capable of quantifying 108–101 CFU of these biovars.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)School of Agricultural and Veterinary Sciences São Paulo State University (FCAV/UNESP)School of Agricultural and Veterinary Sciences São Paulo State University (FCAV/UNESP)Universidade Estadual Paulista (Unesp)Rubio, Marcela da Silva [UNESP]Penha Filho, Rafael Antonio Casarin [UNESP]Almeida, Adriana Maria de [UNESP]Berchieri, Angelo [UNESP]2018-12-11T16:48:19Z2018-12-11T16:48:19Z2017-11-02info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article644-651application/pdfhttp://dx.doi.org/10.1080/03079457.2017.1339866Avian Pathology, v. 46, n. 6, p. 644-651, 2017.1465-33380307-9457http://hdl.handle.net/11449/16993610.1080/03079457.2017.13398662-s2.0-850243930592-s2.0-85024393059.pdfScopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengAvian Pathology0,8710,871info:eu-repo/semantics/openAccess2023-12-31T06:18:32Zoai:repositorio.unesp.br:11449/169936Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-08-05T21:46:50.072663Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false
dc.title.none.fl_str_mv Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum
title Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum
spellingShingle Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum
Rubio, Marcela da Silva [UNESP]
biovar
Fowl typhoid
identification
melting curve
pullorum disease
ROD
SYBr green
title_short Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum
title_full Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum
title_fullStr Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum
title_full_unstemmed Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum
title_sort Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum
author Rubio, Marcela da Silva [UNESP]
author_facet Rubio, Marcela da Silva [UNESP]
Penha Filho, Rafael Antonio Casarin [UNESP]
Almeida, Adriana Maria de [UNESP]
Berchieri, Angelo [UNESP]
author_role author
author2 Penha Filho, Rafael Antonio Casarin [UNESP]
Almeida, Adriana Maria de [UNESP]
Berchieri, Angelo [UNESP]
author2_role author
author
author
dc.contributor.none.fl_str_mv Universidade Estadual Paulista (Unesp)
dc.contributor.author.fl_str_mv Rubio, Marcela da Silva [UNESP]
Penha Filho, Rafael Antonio Casarin [UNESP]
Almeida, Adriana Maria de [UNESP]
Berchieri, Angelo [UNESP]
dc.subject.por.fl_str_mv biovar
Fowl typhoid
identification
melting curve
pullorum disease
ROD
SYBr green
topic biovar
Fowl typhoid
identification
melting curve
pullorum disease
ROD
SYBr green
description Currently there are 2659 Salmonella serovars. The host-specific biovars Salmonella Pullorum and Salmonella Gallinarum cause systemic infections in food-producing and wild birds. Fast diagnosis is crucial to control the dissemination in avian environments. The present work describes the development of a multiplex qPCR in real time using a low-cost DNA dye (SYBr Green) to identify and quantify these biovars. Primers were chosen based on genomic regions of difference (RoD) and optimized to control dimers. Primers pSGP detect both host-specific biovars but not other serovars and pSG and pSP differentiate biovars. Three amplicons showed different melting temperatures (Tm), allowing differentiation. The pSGP amplicon (97 bp) showed Tm of 78°C for both biovars. The pSG amplicon (273 bp) showed a Tm of 86.2°C for S. Gallinarum and pSP amplicon (260 bp) dissociated at 84.8°C for S. Pullorum identification. The multiplex qPCR in real time showed high sensitivity and was capable of quantifying 108–101 CFU of these biovars.
publishDate 2017
dc.date.none.fl_str_mv 2017-11-02
2018-12-11T16:48:19Z
2018-12-11T16:48:19Z
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://dx.doi.org/10.1080/03079457.2017.1339866
Avian Pathology, v. 46, n. 6, p. 644-651, 2017.
1465-3338
0307-9457
http://hdl.handle.net/11449/169936
10.1080/03079457.2017.1339866
2-s2.0-85024393059
2-s2.0-85024393059.pdf
url http://dx.doi.org/10.1080/03079457.2017.1339866
http://hdl.handle.net/11449/169936
identifier_str_mv Avian Pathology, v. 46, n. 6, p. 644-651, 2017.
1465-3338
0307-9457
10.1080/03079457.2017.1339866
2-s2.0-85024393059
2-s2.0-85024393059.pdf
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv Avian Pathology
0,871
0,871
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 644-651
application/pdf
dc.source.none.fl_str_mv Scopus
reponame:Repositório Institucional da UNESP
instname:Universidade Estadual Paulista (UNESP)
instacron:UNESP
instname_str Universidade Estadual Paulista (UNESP)
instacron_str UNESP
institution UNESP
reponame_str Repositório Institucional da UNESP
collection Repositório Institucional da UNESP
repository.name.fl_str_mv Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)
repository.mail.fl_str_mv
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