Impact of extracellular nucleic acids from lactic acid bacteria on qPCR and RT-qPCR results in dairy matrices : implications for defining molecular markers of cell integrity

Autores
Pega, Juan Franco; Descalzo, Adriana Maria; Rossetti, Luciana; Nanni, Mariana; Perez, Carolina Daiana; Rizzo, Sergio Anibal; Diaz, Gabriela Perez
Año de publicación
2017
Idioma
inglés
Tipo de recurso
artículo
Estado
versión aceptada
Descripción
In this study, we evaluated whether quantitative PCR (qPCR) and reverse transcription-qPCR (RT-qPCR) results could be affected by extracellular (free) DNA or RNA, respectively, obtained either from isolated bacteria or from fermented dairy foods manufactured with these microorganisms. These experimental set-ups involved two starter lactic acid bacteria (SLAB), Streptococcus thermophilus and Lactococcus lactis, as models across their most common technological environments: yoghurt, milk and cheese. The presence of free DNA modified qPCR results in levels sufficient to bias SLAB determinations, indicating that DNA may be a poor measure of cellular integrity. Furthermore, we showed for the first time that an order of magnitude more of free RNA than of DNA had to be used to modify RT-qPCR results. Importantly, the interference produced by free DNA and RNA was maintained across all the dairy matrices evaluated, independently of the SLAB and of the source of nucleic acids used, in both negative and positive samples. This study suggests that, contrary to DNA molecules, mRNA from dead microorganisms or from other extracellular sources may not be a determinant factor in skewing molecular quantifications. Therefore, our results support the potential use of mRNA as a molecular signature of cell integrity in dairy matrices.
Inst. de Tecnol. de los Alimentos- ITA
Fil: Pega, Juan Franco. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Descalzo, Adriana Maria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA).LABINTEX-Centre de Coopération Internationale en Recherche Agronomique pour le Déeveloppement (CIRAD), Déepartement PERSYST, UMR Qualisud; Francia
Fil: Rossetti, Luciana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina
Fil: Perez, Carolina Daiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Diaz, Gabriela Perez. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina
Fil: Nanni, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina
Fil: Rizzo, Sergio Anibal. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina
Fuente
LWT - Food science and technology 80 : 416-422. (July 2017)
Materia
Acidos Nucléicos
Bacterias Acidolácticas
Marcadores Genéticos
Productos Lácteos
Nucleic Acids
Lactic Acid Bacteria
Genetic Markers
PCR
Milk Products
Marcadores Moleculares
Reacción en Cadena de la Polimerasa
Nivel de accesibilidad
acceso restringido
Condiciones de uso
Repositorio
INTA Digital (INTA)
Institución
Instituto Nacional de Tecnología Agropecuaria
OAI Identificador
oai:localhost:20.500.12123/1742
Acceder
id INTADig_1b65a53152457ebe9b4ec07ac3ed1d5e
oai_identifier_str oai:localhost:20.500.12123/1742
network_acronym_str INTADig
repository_id_str l
network_name_str INTA Digital (INTA)
spelling Impact of extracellular nucleic acids from lactic acid bacteria on qPCR and RT-qPCR results in dairy matrices : implications for defining molecular markers of cell integrityPega, Juan FrancoDescalzo, Adriana MariaRossetti, LucianaNanni, MarianaPerez, Carolina DaianaRizzo, Sergio AnibalDiaz, Gabriela PerezAcidos NucléicosBacterias AcidolácticasMarcadores GenéticosProductos LácteosNucleic AcidsLactic Acid BacteriaGenetic MarkersPCRMilk ProductsMarcadores MolecularesReacción en Cadena de la PolimerasaIn this study, we evaluated whether quantitative PCR (qPCR) and reverse transcription-qPCR (RT-qPCR) results could be affected by extracellular (free) DNA or RNA, respectively, obtained either from isolated bacteria or from fermented dairy foods manufactured with these microorganisms. These experimental set-ups involved two starter lactic acid bacteria (SLAB), Streptococcus thermophilus and Lactococcus lactis, as models across their most common technological environments: yoghurt, milk and cheese. The presence of free DNA modified qPCR results in levels sufficient to bias SLAB determinations, indicating that DNA may be a poor measure of cellular integrity. Furthermore, we showed for the first time that an order of magnitude more of free RNA than of DNA had to be used to modify RT-qPCR results. Importantly, the interference produced by free DNA and RNA was maintained across all the dairy matrices evaluated, independently of the SLAB and of the source of nucleic acids used, in both negative and positive samples. This study suggests that, contrary to DNA molecules, mRNA from dead microorganisms or from other extracellular sources may not be a determinant factor in skewing molecular quantifications. Therefore, our results support the potential use of mRNA as a molecular signature of cell integrity in dairy matrices.Inst. de Tecnol. de los Alimentos- ITAFil: Pega, Juan Franco. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Descalzo, Adriana Maria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA).LABINTEX-Centre de Coopération Internationale en Recherche Agronomique pour le Déeveloppement (CIRAD), Déepartement PERSYST, UMR Qualisud; FranciaFil: Rossetti, Luciana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; ArgentinaFil: Perez, Carolina Daiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Diaz, Gabriela Perez. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; ArgentinaFil: Nanni, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; ArgentinaFil: Rizzo, Sergio Anibal. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina2017-11-10T12:07:47Z2017-11-10T12:07:47Z2017-07info:eu-repo/semantics/articleinfo:eu-repo/semantics/acceptedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttp://hdl.handle.net/20.500.12123/1742https://www.sciencedirect.com/science/article/pii/S002364381730155X0023-6438https://doi.org/10.1016/j.lwt.2017.03.010LWT - Food science and technology 80 : 416-422. (July 2017)reponame:INTA Digital (INTA)instname:Instituto Nacional de Tecnología Agropecuariaenginfo:eu-repo/semantics/restrictedAccess2025-10-30T11:22:38Zoai:localhost:20.500.12123/1742instacron:INTAInstitucionalhttp://repositorio.inta.gob.ar/Organismo científico-tecnológicoNo correspondehttp://repositorio.inta.gob.ar/oai/requesttripaldi.nicolas@inta.gob.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:l2025-10-30 11:22:39.042INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse
dc.title.none.fl_str_mv Impact of extracellular nucleic acids from lactic acid bacteria on qPCR and RT-qPCR results in dairy matrices : implications for defining molecular markers of cell integrity
title Impact of extracellular nucleic acids from lactic acid bacteria on qPCR and RT-qPCR results in dairy matrices : implications for defining molecular markers of cell integrity
spellingShingle Impact of extracellular nucleic acids from lactic acid bacteria on qPCR and RT-qPCR results in dairy matrices : implications for defining molecular markers of cell integrity
Pega, Juan Franco
Acidos Nucléicos
Bacterias Acidolácticas
Marcadores Genéticos
Productos Lácteos
Nucleic Acids
Lactic Acid Bacteria
Genetic Markers
PCR
Milk Products
Marcadores Moleculares
Reacción en Cadena de la Polimerasa
title_short Impact of extracellular nucleic acids from lactic acid bacteria on qPCR and RT-qPCR results in dairy matrices : implications for defining molecular markers of cell integrity
title_full Impact of extracellular nucleic acids from lactic acid bacteria on qPCR and RT-qPCR results in dairy matrices : implications for defining molecular markers of cell integrity
title_fullStr Impact of extracellular nucleic acids from lactic acid bacteria on qPCR and RT-qPCR results in dairy matrices : implications for defining molecular markers of cell integrity
title_full_unstemmed Impact of extracellular nucleic acids from lactic acid bacteria on qPCR and RT-qPCR results in dairy matrices : implications for defining molecular markers of cell integrity
title_sort Impact of extracellular nucleic acids from lactic acid bacteria on qPCR and RT-qPCR results in dairy matrices : implications for defining molecular markers of cell integrity
dc.creator.none.fl_str_mv Pega, Juan Franco
Descalzo, Adriana Maria
Rossetti, Luciana
Nanni, Mariana
Perez, Carolina Daiana
Rizzo, Sergio Anibal
Diaz, Gabriela Perez
author Pega, Juan Franco
author_facet Pega, Juan Franco
Descalzo, Adriana Maria
Rossetti, Luciana
Nanni, Mariana
Perez, Carolina Daiana
Rizzo, Sergio Anibal
Diaz, Gabriela Perez
author_role author
author2 Descalzo, Adriana Maria
Rossetti, Luciana
Nanni, Mariana
Perez, Carolina Daiana
Rizzo, Sergio Anibal
Diaz, Gabriela Perez
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv Acidos Nucléicos
Bacterias Acidolácticas
Marcadores Genéticos
Productos Lácteos
Nucleic Acids
Lactic Acid Bacteria
Genetic Markers
PCR
Milk Products
Marcadores Moleculares
Reacción en Cadena de la Polimerasa
topic Acidos Nucléicos
Bacterias Acidolácticas
Marcadores Genéticos
Productos Lácteos
Nucleic Acids
Lactic Acid Bacteria
Genetic Markers
PCR
Milk Products
Marcadores Moleculares
Reacción en Cadena de la Polimerasa
dc.description.none.fl_txt_mv In this study, we evaluated whether quantitative PCR (qPCR) and reverse transcription-qPCR (RT-qPCR) results could be affected by extracellular (free) DNA or RNA, respectively, obtained either from isolated bacteria or from fermented dairy foods manufactured with these microorganisms. These experimental set-ups involved two starter lactic acid bacteria (SLAB), Streptococcus thermophilus and Lactococcus lactis, as models across their most common technological environments: yoghurt, milk and cheese. The presence of free DNA modified qPCR results in levels sufficient to bias SLAB determinations, indicating that DNA may be a poor measure of cellular integrity. Furthermore, we showed for the first time that an order of magnitude more of free RNA than of DNA had to be used to modify RT-qPCR results. Importantly, the interference produced by free DNA and RNA was maintained across all the dairy matrices evaluated, independently of the SLAB and of the source of nucleic acids used, in both negative and positive samples. This study suggests that, contrary to DNA molecules, mRNA from dead microorganisms or from other extracellular sources may not be a determinant factor in skewing molecular quantifications. Therefore, our results support the potential use of mRNA as a molecular signature of cell integrity in dairy matrices.
Inst. de Tecnol. de los Alimentos- ITA
Fil: Pega, Juan Franco. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Descalzo, Adriana Maria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA).LABINTEX-Centre de Coopération Internationale en Recherche Agronomique pour le Déeveloppement (CIRAD), Déepartement PERSYST, UMR Qualisud; Francia
Fil: Rossetti, Luciana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina
Fil: Perez, Carolina Daiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Diaz, Gabriela Perez. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina
Fil: Nanni, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina
Fil: Rizzo, Sergio Anibal. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina
description In this study, we evaluated whether quantitative PCR (qPCR) and reverse transcription-qPCR (RT-qPCR) results could be affected by extracellular (free) DNA or RNA, respectively, obtained either from isolated bacteria or from fermented dairy foods manufactured with these microorganisms. These experimental set-ups involved two starter lactic acid bacteria (SLAB), Streptococcus thermophilus and Lactococcus lactis, as models across their most common technological environments: yoghurt, milk and cheese. The presence of free DNA modified qPCR results in levels sufficient to bias SLAB determinations, indicating that DNA may be a poor measure of cellular integrity. Furthermore, we showed for the first time that an order of magnitude more of free RNA than of DNA had to be used to modify RT-qPCR results. Importantly, the interference produced by free DNA and RNA was maintained across all the dairy matrices evaluated, independently of the SLAB and of the source of nucleic acids used, in both negative and positive samples. This study suggests that, contrary to DNA molecules, mRNA from dead microorganisms or from other extracellular sources may not be a determinant factor in skewing molecular quantifications. Therefore, our results support the potential use of mRNA as a molecular signature of cell integrity in dairy matrices.
publishDate 2017
dc.date.none.fl_str_mv 2017-11-10T12:07:47Z
2017-11-10T12:07:47Z
2017-07
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/acceptedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str acceptedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/20.500.12123/1742
https://www.sciencedirect.com/science/article/pii/S002364381730155X
0023-6438
https://doi.org/10.1016/j.lwt.2017.03.010
url http://hdl.handle.net/20.500.12123/1742
https://www.sciencedirect.com/science/article/pii/S002364381730155X
https://doi.org/10.1016/j.lwt.2017.03.010
identifier_str_mv 0023-6438
dc.language.none.fl_str_mv eng
language eng
dc.rights.none.fl_str_mv info:eu-repo/semantics/restrictedAccess
eu_rights_str_mv restrictedAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv LWT - Food science and technology 80 : 416-422. (July 2017)
reponame:INTA Digital (INTA)
instname:Instituto Nacional de Tecnología Agropecuaria
reponame_str INTA Digital (INTA)
collection INTA Digital (INTA)
instname_str Instituto Nacional de Tecnología Agropecuaria
repository.name.fl_str_mv INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuaria
repository.mail.fl_str_mv tripaldi.nicolas@inta.gob.ar
_version_ 1847419423869108224
score 12.589754

Publicaciones similares