Design and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitis

Objective: This study aimed to standardize a quantitative polymerase chain reaction (qPCR)-based test to identify and quantify the uncultivable bacteria associated with periodontitis. Methods: The standardization of qPCR, the curves for the quantification of Eubacterium saphenum, Eubacterium brachy,...

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Autores:
Castillo, Yormaris
Delgadillo, Nathaly Andrea
Neuta, Yineth
Iniesta, Margarita
Sanz, Mariano
Herrera, David
Pianeta, Roquelina
Lafaurie, Gloria Inés
Castillo, Diana Marcela
Tipo de recurso:
Article of journal
Fecha de publicación:
2023
Institución:
Universidad El Bosque
Repositorio:
Repositorio U. El Bosque
Idioma:
eng
OAI Identifier:
oai:repositorio.unbosque.edu.co:20.500.12495/11082
Acceso en línea:
http://hdl.handle.net/20.500.12495/11082
https://doi.org/10.1016/j.archoralbio.2023.105758
Palabra clave:
Eficacia de la PCR
QPCR
Número de copias
Bacterias orales
Periodontitis
PCR efficiency
QPCR
Copy number
Oral bacteria
Periodontitis
Rights
openAccess
License
Attribution-NonCommercial-NoDerivatives 4.0 Internacional
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dc.title.spa.fl_str_mv Design and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitis
dc.title.translated.spa.fl_str_mv Design and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitis
title Design and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitis
spellingShingle Design and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitis
Eficacia de la PCR
QPCR
Número de copias
Bacterias orales
Periodontitis
PCR efficiency
QPCR
Copy number
Oral bacteria
Periodontitis
title_short Design and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitis
title_full Design and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitis
title_fullStr Design and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitis
title_full_unstemmed Design and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitis
title_sort Design and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitis
dc.creator.fl_str_mv Castillo, Yormaris
Delgadillo, Nathaly Andrea
Neuta, Yineth
Iniesta, Margarita
Sanz, Mariano
Herrera, David
Pianeta, Roquelina
Lafaurie, Gloria Inés
Castillo, Diana Marcela
dc.contributor.author.none.fl_str_mv Castillo, Yormaris
Delgadillo, Nathaly Andrea
Neuta, Yineth
Iniesta, Margarita
Sanz, Mariano
Herrera, David
Pianeta, Roquelina
Lafaurie, Gloria Inés
Castillo, Diana Marcela
dc.contributor.orcid.none.fl_str_mv Castillo, Yormaris [0000-0002-8673-8449]
Delgadillo, Nathaly Andrea [0000-0001-8842-549X]
Neuta, Yineth [0000-0001-9350-0941]
Iniesta, Margarita [0000-0001-7557-4023]
dc.subject.spa.fl_str_mv Eficacia de la PCR
QPCR
Número de copias
Bacterias orales
Periodontitis
topic Eficacia de la PCR
QPCR
Número de copias
Bacterias orales
Periodontitis
PCR efficiency
QPCR
Copy number
Oral bacteria
Periodontitis
dc.subject.keywords.spa.fl_str_mv PCR efficiency
QPCR
Copy number
Oral bacteria
Periodontitis
description Objective: This study aimed to standardize a quantitative polymerase chain reaction (qPCR)-based test to identify and quantify the uncultivable bacteria associated with periodontitis. Methods: The standardization of qPCR, the curves for the quantification of Eubacterium saphenum, Eubacterium brachy, Desulfobulbus oralis, and Filifactor alocis were developed by cloning the 16 S rRNA target gene fragment, using the GEMTEasy vector. The qPCRs were validated in 55 subgingival biofilm clinical samples, from different stages of periodontitis and from periodontally healthy/gingivitis individuals, which were previously evaluated by next-generation sequencing (NGS). The results obtained by the two methods were compared by the concordance of Cohen's Kappa index, and sensitivity, specificity, receiver operating characteristic (ROC) curve, and predictive values were established. Results: obtained by the two methods were compared using the concordance of Cohen's Kappa index, and sensitivity, specificity, predictive values, and ROC curves were generated. The qPCR test was standardized with efficiencies between 90% and 100% and R2: 0.997–0.999. Concordance between the qPCR and NSG was moderate to F. alocis (agreement 78.2%; kappa 0.56, p < 0.05) and fair to the other microorganisms (agreement 67.27%−72.73; kappa 0.37–0.38, p < 0.05). qPCR exhibited a high sensitivity (82.2–100%) and specificity (100%) for E. brachy, E. saphenum, and F. alocis. Sensitivity was lower to D. oralis. Conversely, qPCR demonstrated higher sensitivity to E. saphenum than NSG (100 vs. 68.1). Conclusions: The uncultivable microorganisms associated with periodontitis, D. oralis, E. brachy, E. saphenum, and F. alocis can be detected and quantified with the newly developed and validates qPCR test.
publishDate 2023
dc.date.accessioned.none.fl_str_mv 2023-07-19T16:32:13Z
dc.date.available.none.fl_str_mv 2023-07-19T16:32:13Z
dc.date.issued.none.fl_str_mv 2023
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dc.type.local.none.fl_str_mv Artículo de revista
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dc.identifier.issn.none.fl_str_mv 0003-9969
dc.identifier.uri.none.fl_str_mv http://hdl.handle.net/20.500.12495/11082
dc.identifier.doi.none.fl_str_mv https://doi.org/10.1016/j.archoralbio.2023.105758
dc.identifier.instname.spa.fl_str_mv instname:Universidad El Bosque
dc.identifier.reponame.spa.fl_str_mv reponame:Repositorio Institucional Universidad El Bosque
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identifier_str_mv 0003-9969
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url http://hdl.handle.net/20.500.12495/11082
https://doi.org/10.1016/j.archoralbio.2023.105758
dc.language.iso.none.fl_str_mv eng
language eng
dc.relation.ispartofseries.spa.fl_str_mv Archives of Oral Biology, 0003-9969, 154, 2023, 1 - 8
dc.relation.uri.none.fl_str_mv https://www.sciencedirect.com/science/article/pii/S0003996923001462?pes=vor
dc.rights.*.fl_str_mv Attribution-NonCommercial-NoDerivatives 4.0 Internacional
dc.rights.uri.*.fl_str_mv http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.rights.local.spa.fl_str_mv Acceso abierto
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eu_rights_str_mv openAccess
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dc.publisher.spa.fl_str_mv Elsevier
dc.publisher.journal.spa.fl_str_mv Archives of Oral Biology
institution Universidad El Bosque
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spelling Castillo, YormarisDelgadillo, Nathaly AndreaNeuta, YinethIniesta, MargaritaSanz, MarianoHerrera, DavidPianeta, RoquelinaLafaurie, Gloria InésCastillo, Diana MarcelaCastillo, Yormaris [0000-0002-8673-8449]Delgadillo, Nathaly Andrea [0000-0001-8842-549X]Neuta, Yineth [0000-0001-9350-0941]Iniesta, Margarita [0000-0001-7557-4023]2023-07-19T16:32:13Z2023-07-19T16:32:13Z20230003-9969http://hdl.handle.net/20.500.12495/11082https://doi.org/10.1016/j.archoralbio.2023.105758instname:Universidad El Bosquereponame:Repositorio Institucional Universidad El Bosquerepourl:https://repositorio.unbosque.edu.coapplication/pdfengElsevierArchives of Oral BiologyArchives of Oral Biology, 0003-9969, 154, 2023, 1 - 8https://www.sciencedirect.com/science/article/pii/S0003996923001462?pes=vorAttribution-NonCommercial-NoDerivatives 4.0 Internacionalhttp://creativecommons.org/licenses/by-nc-nd/4.0/Acceso abiertoinfo:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Eficacia de la PCRQPCRNúmero de copiasBacterias oralesPeriodontitisPCR efficiencyQPCRCopy numberOral bacteriaPeriodontitisDesign and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitisDesign and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitisArtículo de revistainfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501http://purl.org/coar/resource_type/c_2df8fbb1info:eu-repo/semantics/articlehttp://purl.org/coar/version/c_970fb48d4fbd8a85Objective: This study aimed to standardize a quantitative polymerase chain reaction (qPCR)-based test to identify and quantify the uncultivable bacteria associated with periodontitis. Methods: The standardization of qPCR, the curves for the quantification of Eubacterium saphenum, Eubacterium brachy, Desulfobulbus oralis, and Filifactor alocis were developed by cloning the 16 S rRNA target gene fragment, using the GEMTEasy vector. The qPCRs were validated in 55 subgingival biofilm clinical samples, from different stages of periodontitis and from periodontally healthy/gingivitis individuals, which were previously evaluated by next-generation sequencing (NGS). The results obtained by the two methods were compared by the concordance of Cohen's Kappa index, and sensitivity, specificity, receiver operating characteristic (ROC) curve, and predictive values were established. Results: obtained by the two methods were compared using the concordance of Cohen's Kappa index, and sensitivity, specificity, predictive values, and ROC curves were generated. The qPCR test was standardized with efficiencies between 90% and 100% and R2: 0.997–0.999. Concordance between the qPCR and NSG was moderate to F. alocis (agreement 78.2%; kappa 0.56, p < 0.05) and fair to the other microorganisms (agreement 67.27%−72.73; kappa 0.37–0.38, p < 0.05). qPCR exhibited a high sensitivity (82.2–100%) and specificity (100%) for E. brachy, E. saphenum, and F. alocis. Sensitivity was lower to D. oralis. Conversely, qPCR demonstrated higher sensitivity to E. saphenum than NSG (100 vs. 68.1). Conclusions: The uncultivable microorganisms associated with periodontitis, D. oralis, E. brachy, E. saphenum, and F. alocis can be detected and quantified with the newly developed and validates qPCR test.ORIGINAL1-s2.0-S0003996923001462-main.pdf1-s2.0-S0003996923001462-main.pdfDesign and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitisapplication/pdf2994222https://repositorio.unbosque.edu.co/bitstreams/7b0eb293-5fe8-48da-afa3-1333803c0ec4/downloadc0b584a202fb262095b0a590ffdb7de5MD51CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8908https://repositorio.unbosque.edu.co/bitstreams/974b9aa6-1668-497c-91a8-79e918457fa0/download0175ea4a2d4caec4bbcc37e300941108MD52LICENSElicense.txtlicense.txttext/plain; charset=utf-82000https://repositorio.unbosque.edu.co/bitstreams/1b361c0b-3d26-42d7-bb42-1968548304c8/download17cc15b951e7cc6b3728a574117320f9MD53THUMBNAIL1-s2.0-S0003996923001462-main.pdf.jpg1-s2.0-S0003996923001462-main.pdf.jpgIM Thumbnailimage/jpeg10064https://repositorio.unbosque.edu.co/bitstreams/5d942738-acd9-42b8-a3ad-b45f8137158f/downloadcf0d4d5306632d89a9b77a8064adb748MD54TEXT1-s2.0-S0003996923001462-main.pdf.txt1-s2.0-S0003996923001462-main.pdf.txtExtracted texttext/plain49856https://repositorio.unbosque.edu.co/bitstreams/4c0bb448-473c-419f-9396-d5bb06cb159d/downloadc4ddfe82e25580e3b754619c737af738MD5520.500.12495/11082oai:repositorio.unbosque.edu.co:20.500.12495/110822024-02-07 07:44:21.896http://creativecommons.org/licenses/by-nc-nd/4.0/Attribution-NonCommercial-NoDerivatives 4.0 Internacionalopen.accesshttps://repositorio.unbosque.edu.coRepositorio Institucional Universidad El Bosquebibliotecas@biteca.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