A novel oligonucleotide pair for genotyping members of the pseudomonas genus by single-round PCR amplification of the gyrb gene

Autores
Agaras, Betina Cecilia; Valverde, Claudio Fabián
Año de publicación
2018
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Pseudomonas is a phylogenetically diverse bacterial genus which is broadly distributed in different ecological niches, and whose taxonomy is continuously under revision. For that purpose, gyrB is one of the housekeeping genes routinely used for multilocus sequence analysis (MLSA). As we noticed that there was not a single primer pair available in the literature suitable for direct sequencing of this gene, we decided to design a unique oligonucleotide pair and to set up a polymerase chain reaction (PCR) protocol to obtain a single amplicon for the entire Pseudomonas genus. Based on the available gyrB sequence from 148 Pseudomonas species, we identified highly conserved regions to design oligonucleotides without fully degenerate positions. We then set up cycling conditions for achieving high specificity and yield of the PCR protocol. Then, we showed that the amplicons produced with this procedure were appropriate for direct sequencing with both primers, obtaining more than 95% of amplicons coverage. Finally, we demonstrated that a PCR-RFLP (restriction fragment length polymorphism) approach served to differentiate among Pseudomonas species, and even between members of the same species.
Fil: Agaras, Betina Cecilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Materia
GYRB
MLSA
PCR-RFLP
PSEUDOMONAS
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/162369
Acceder
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network_name_str CONICET Digital (CONICET)
spelling A novel oligonucleotide pair for genotyping members of the pseudomonas genus by single-round PCR amplification of the gyrb geneAgaras, Betina CeciliaValverde, Claudio FabiánGYRBMLSAPCR-RFLPPSEUDOMONAShttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Pseudomonas is a phylogenetically diverse bacterial genus which is broadly distributed in different ecological niches, and whose taxonomy is continuously under revision. For that purpose, gyrB is one of the housekeeping genes routinely used for multilocus sequence analysis (MLSA). As we noticed that there was not a single primer pair available in the literature suitable for direct sequencing of this gene, we decided to design a unique oligonucleotide pair and to set up a polymerase chain reaction (PCR) protocol to obtain a single amplicon for the entire Pseudomonas genus. Based on the available gyrB sequence from 148 Pseudomonas species, we identified highly conserved regions to design oligonucleotides without fully degenerate positions. We then set up cycling conditions for achieving high specificity and yield of the PCR protocol. Then, we showed that the amplicons produced with this procedure were appropriate for direct sequencing with both primers, obtaining more than 95% of amplicons coverage. Finally, we demonstrated that a PCR-RFLP (restriction fragment length polymorphism) approach served to differentiate among Pseudomonas species, and even between members of the same species.Fil: Agaras, Betina Cecilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaMDPI AG2018-07info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/162369Agaras, Betina Cecilia; Valverde, Claudio Fabián; A novel oligonucleotide pair for genotyping members of the pseudomonas genus by single-round PCR amplification of the gyrb gene; MDPI AG; Methods and Protocols; 1; 3; 7-2018; 1-132409-9279CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.mdpi.com/2409-9279/1/3/24info:eu-repo/semantics/altIdentifier/doi/10.3390/mps1030024info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:26:51Zoai:ri.conicet.gov.ar:11336/162369instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-22 11:26:51.325CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv A novel oligonucleotide pair for genotyping members of the pseudomonas genus by single-round PCR amplification of the gyrb gene
title A novel oligonucleotide pair for genotyping members of the pseudomonas genus by single-round PCR amplification of the gyrb gene
spellingShingle A novel oligonucleotide pair for genotyping members of the pseudomonas genus by single-round PCR amplification of the gyrb gene
Agaras, Betina Cecilia
GYRB
MLSA
PCR-RFLP
PSEUDOMONAS
title_short A novel oligonucleotide pair for genotyping members of the pseudomonas genus by single-round PCR amplification of the gyrb gene
title_full A novel oligonucleotide pair for genotyping members of the pseudomonas genus by single-round PCR amplification of the gyrb gene
title_fullStr A novel oligonucleotide pair for genotyping members of the pseudomonas genus by single-round PCR amplification of the gyrb gene
title_full_unstemmed A novel oligonucleotide pair for genotyping members of the pseudomonas genus by single-round PCR amplification of the gyrb gene
title_sort A novel oligonucleotide pair for genotyping members of the pseudomonas genus by single-round PCR amplification of the gyrb gene
dc.creator.none.fl_str_mv Agaras, Betina Cecilia
Valverde, Claudio Fabián
author Agaras, Betina Cecilia
author_facet Agaras, Betina Cecilia
Valverde, Claudio Fabián
author_role author
author2 Valverde, Claudio Fabián
author2_role author
dc.subject.none.fl_str_mv GYRB
MLSA
PCR-RFLP
PSEUDOMONAS
topic GYRB
MLSA
PCR-RFLP
PSEUDOMONAS
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Pseudomonas is a phylogenetically diverse bacterial genus which is broadly distributed in different ecological niches, and whose taxonomy is continuously under revision. For that purpose, gyrB is one of the housekeeping genes routinely used for multilocus sequence analysis (MLSA). As we noticed that there was not a single primer pair available in the literature suitable for direct sequencing of this gene, we decided to design a unique oligonucleotide pair and to set up a polymerase chain reaction (PCR) protocol to obtain a single amplicon for the entire Pseudomonas genus. Based on the available gyrB sequence from 148 Pseudomonas species, we identified highly conserved regions to design oligonucleotides without fully degenerate positions. We then set up cycling conditions for achieving high specificity and yield of the PCR protocol. Then, we showed that the amplicons produced with this procedure were appropriate for direct sequencing with both primers, obtaining more than 95% of amplicons coverage. Finally, we demonstrated that a PCR-RFLP (restriction fragment length polymorphism) approach served to differentiate among Pseudomonas species, and even between members of the same species.
Fil: Agaras, Betina Cecilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
description Pseudomonas is a phylogenetically diverse bacterial genus which is broadly distributed in different ecological niches, and whose taxonomy is continuously under revision. For that purpose, gyrB is one of the housekeeping genes routinely used for multilocus sequence analysis (MLSA). As we noticed that there was not a single primer pair available in the literature suitable for direct sequencing of this gene, we decided to design a unique oligonucleotide pair and to set up a polymerase chain reaction (PCR) protocol to obtain a single amplicon for the entire Pseudomonas genus. Based on the available gyrB sequence from 148 Pseudomonas species, we identified highly conserved regions to design oligonucleotides without fully degenerate positions. We then set up cycling conditions for achieving high specificity and yield of the PCR protocol. Then, we showed that the amplicons produced with this procedure were appropriate for direct sequencing with both primers, obtaining more than 95% of amplicons coverage. Finally, we demonstrated that a PCR-RFLP (restriction fragment length polymorphism) approach served to differentiate among Pseudomonas species, and even between members of the same species.
publishDate 2018
dc.date.none.fl_str_mv 2018-07
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/162369
Agaras, Betina Cecilia; Valverde, Claudio Fabián; A novel oligonucleotide pair for genotyping members of the pseudomonas genus by single-round PCR amplification of the gyrb gene; MDPI AG; Methods and Protocols; 1; 3; 7-2018; 1-13
2409-9279
CONICET Digital
CONICET
url http://hdl.handle.net/11336/162369
identifier_str_mv Agaras, Betina Cecilia; Valverde, Claudio Fabián; A novel oligonucleotide pair for genotyping members of the pseudomonas genus by single-round PCR amplification of the gyrb gene; MDPI AG; Methods and Protocols; 1; 3; 7-2018; 1-13
2409-9279
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.mdpi.com/2409-9279/1/3/24
info:eu-repo/semantics/altIdentifier/doi/10.3390/mps1030024
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv MDPI AG
publisher.none.fl_str_mv MDPI AG
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 12.982451

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