Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays

Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commerci...

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Autores: Abras Feliu, Alba, Ballart, Cristina, Llovet, Teresa, Roig, Carme, Gutiérrez, Cristina, Tebar, Sílvia, Berenguer, Pere, Pinazo, María-Jesús, Posada, Elizabeth, Gascón i Brustenga, Joaquim, Schijman, Alejandro G., Gállego, Montserrat, Muñoz, Carmen
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2018
País:España
Institución:Varias* (Consorci de Biblioteques Universitáries de Catalunya, Centre de Serveis Científics i Acadèmics de Catalunya)
Repositorio:Recercat. Dipósit de la Recerca de Catalunya
OAI Identifier:oai:recercat.cat:10256/16232
Acceso en línea:http://hdl.handle.net/10256/16232
Access Level:acceso abierto
Palabra clave:ADN -- Anàlisi
DNA -- Analysis
Diagnòstic molecular
Molecular diagnosis
Biologia molecular
Molecular biology
Tripanosoma cruzi -- Genètica
Tripanosoma cruzi -- Genetics
Chagas, Malaltia de -- Aspectes genètics
Chagas' disease -- Genetic aspects
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spelling Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assaysAbras Feliu, AlbaBallart, CristinaLlovet, TeresaRoig, CarmeGutiérrez, CristinaTebar, SílviaBerenguer, PerePinazo, María-JesúsPosada, ElizabethGascón i Brustenga, JoaquimSchijman, Alejandro G.Gállego, MontserratMuñoz, CarmenADN -- AnàlisiDNA -- AnalysisDiagnòstic molecularMolecular diagnosisBiologia molecularMolecular biologyTripanosoma cruzi -- GenèticaTripanosoma cruzi -- GeneticsChagas, Malaltia de -- Aspectes genèticsChagas' disease -- Genetic aspectsPolymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process. Methodology/Principal findings We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately. Conclusions/Significance This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centersPublic Library of Science (PLoS)2018info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionpeer-reviewedapplication/pdfhttp://hdl.handle.net/10256/16232PLoS ONE, 2018, vol. 13, núm. 4, p. e0195738Articles publicats (D-B)reponame:Recercat. Dipósit de la Recerca de Catalunyainstname:Varias* (Consorci de Biblioteques Universitáries de Catalunya, Centre de Serveis Científics i Acadèmics de Catalunya)Inglésinfo:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pone.0195738info:eu-repo/semantics/altIdentifier/eissn/1932-6203Attribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:recercat.cat:10256/162322026-05-29T05:05:01Z
dc.title.none.fl_str_mv Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
title Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
spellingShingle Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
Abras Feliu, Alba
ADN -- Anàlisi
DNA -- Analysis
Diagnòstic molecular
Molecular diagnosis
Biologia molecular
Molecular biology
Tripanosoma cruzi -- Genètica
Tripanosoma cruzi -- Genetics
Chagas, Malaltia de -- Aspectes genètics
Chagas' disease -- Genetic aspects
title_short Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
title_full Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
title_fullStr Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
title_full_unstemmed Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
title_sort Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
dc.creator.none.fl_str_mv Abras Feliu, Alba
Ballart, Cristina
Llovet, Teresa
Roig, Carme
Gutiérrez, Cristina
Tebar, Sílvia
Berenguer, Pere
Pinazo, María-Jesús
Posada, Elizabeth
Gascón i Brustenga, Joaquim
Schijman, Alejandro G.
Gállego, Montserrat
Muñoz, Carmen
author Abras Feliu, Alba
author_facet Abras Feliu, Alba
Ballart, Cristina
Llovet, Teresa
Roig, Carme
Gutiérrez, Cristina
Tebar, Sílvia
Berenguer, Pere
Pinazo, María-Jesús
Posada, Elizabeth
Gascón i Brustenga, Joaquim
Schijman, Alejandro G.
Gállego, Montserrat
Muñoz, Carmen
author_role author
author2 Ballart, Cristina
Llovet, Teresa
Roig, Carme
Gutiérrez, Cristina
Tebar, Sílvia
Berenguer, Pere
Pinazo, María-Jesús
Posada, Elizabeth
Gascón i Brustenga, Joaquim
Schijman, Alejandro G.
Gállego, Montserrat
Muñoz, Carmen
author2_role author
author
author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv ADN -- Anàlisi
DNA -- Analysis
Diagnòstic molecular
Molecular diagnosis
Biologia molecular
Molecular biology
Tripanosoma cruzi -- Genètica
Tripanosoma cruzi -- Genetics
Chagas, Malaltia de -- Aspectes genètics
Chagas' disease -- Genetic aspects
topic ADN -- Anàlisi
DNA -- Analysis
Diagnòstic molecular
Molecular diagnosis
Biologia molecular
Molecular biology
Tripanosoma cruzi -- Genètica
Tripanosoma cruzi -- Genetics
Chagas, Malaltia de -- Aspectes genètics
Chagas' disease -- Genetic aspects
description Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process. Methodology/Principal findings We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately. Conclusions/Significance This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers
publishDate 2018
dc.date.none.fl_str_mv 2018
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
peer-reviewed
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10256/16232
url http://hdl.handle.net/10256/16232
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pone.0195738
info:eu-repo/semantics/altIdentifier/eissn/1932-6203
dc.rights.none.fl_str_mv Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Public Library of Science (PLoS)
publisher.none.fl_str_mv Public Library of Science (PLoS)
dc.source.none.fl_str_mv PLoS ONE, 2018, vol. 13, núm. 4, p. e0195738
Articles publicats (D-B)
reponame:Recercat. Dipósit de la Recerca de Catalunya
instname:Varias* (Consorci de Biblioteques Universitáries de Catalunya, Centre de Serveis Científics i Acadèmics de Catalunya)
instname_str Varias* (Consorci de Biblioteques Universitáries de Catalunya, Centre de Serveis Científics i Acadèmics de Catalunya)
reponame_str Recercat. Dipósit de la Recerca de Catalunya
collection Recercat. Dipósit de la Recerca de Catalunya
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15.812429

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