Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples.
Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivit...
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doaj-bb5bcd95af574c5fbbcff0a48941e69a2020-11-25T01:44:58ZengPublic Library of Science (PLoS)PLoS Neglected Tropical Diseases1935-27271935-27352015-05-0195e000376510.1371/journal.pntd.0003765Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples.Carolina I CuraTomas DuffyRaúl H LuceroMargarita BisioJulie PéneauMatilde Jimenez-CoelloEva CalabuigMaría J GimenezEdward Valencia AyalaSonia A KjosJosé SantallaSusan M MahaneyNelly M CayoClaudia NagelLaura BarcánEdith S Málaga MachacaKarla Y Acosta VianaLaurent BrutusSusana B OcampoChristine AznarCesar A Cuba CubaRicardo E GürtlerJanine M RamseyIsabela RibeiroJohn L VandeBergZaida E YadonAntonio OsunaAlejandro G SchijmanTrypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.http://europepmc.org/articles/PMC4437652?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Carolina I Cura Tomas Duffy Raúl H Lucero Margarita Bisio Julie Péneau Matilde Jimenez-Coello Eva Calabuig María J Gimenez Edward Valencia Ayala Sonia A Kjos José Santalla Susan M Mahaney Nelly M Cayo Claudia Nagel Laura Barcán Edith S Málaga Machaca Karla Y Acosta Viana Laurent Brutus Susana B Ocampo Christine Aznar Cesar A Cuba Cuba Ricardo E Gürtler Janine M Ramsey Isabela Ribeiro John L VandeBerg Zaida E Yadon Antonio Osuna Alejandro G Schijman |
spellingShingle |
Carolina I Cura Tomas Duffy Raúl H Lucero Margarita Bisio Julie Péneau Matilde Jimenez-Coello Eva Calabuig María J Gimenez Edward Valencia Ayala Sonia A Kjos José Santalla Susan M Mahaney Nelly M Cayo Claudia Nagel Laura Barcán Edith S Málaga Machaca Karla Y Acosta Viana Laurent Brutus Susana B Ocampo Christine Aznar Cesar A Cuba Cuba Ricardo E Gürtler Janine M Ramsey Isabela Ribeiro John L VandeBerg Zaida E Yadon Antonio Osuna Alejandro G Schijman Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples. PLoS Neglected Tropical Diseases |
author_facet |
Carolina I Cura Tomas Duffy Raúl H Lucero Margarita Bisio Julie Péneau Matilde Jimenez-Coello Eva Calabuig María J Gimenez Edward Valencia Ayala Sonia A Kjos José Santalla Susan M Mahaney Nelly M Cayo Claudia Nagel Laura Barcán Edith S Málaga Machaca Karla Y Acosta Viana Laurent Brutus Susana B Ocampo Christine Aznar Cesar A Cuba Cuba Ricardo E Gürtler Janine M Ramsey Isabela Ribeiro John L VandeBerg Zaida E Yadon Antonio Osuna Alejandro G Schijman |
author_sort |
Carolina I Cura |
title |
Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples. |
title_short |
Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples. |
title_full |
Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples. |
title_fullStr |
Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples. |
title_full_unstemmed |
Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples. |
title_sort |
multiplex real-time pcr assay using taqman probes for the identification of trypanosoma cruzi dtus in biological and clinical samples. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS Neglected Tropical Diseases |
issn |
1935-2727 1935-2735 |
publishDate |
2015-05-01 |
description |
Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. |
url |
http://europepmc.org/articles/PMC4437652?pdf=render |
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