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Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

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dc.contributor.author Cura, Carolina I.
dc.contributor.author Duffy, Tomas
dc.contributor.author Lucero, Raul H.
dc.contributor.author Bisio, Margarita
dc.contributor.author Peneau, Julie
dc.contributor.author Jimenez-Coello, Matilde
dc.contributor.author Calabuig, Eva
dc.contributor.author Gimenez, Maria J.
dc.contributor.author Valencia Ayala, Edward
dc.contributor.author Kjos, Sonia A.
dc.contributor.author Santalla, Jose
dc.contributor.author Mahaney, Susan M.
dc.contributor.author Cayo, Nelly M.
dc.contributor.author Nagel, Claudia
dc.contributor.author Barcan, Laura
dc.contributor.author Malaga Machaca, Edith S.
dc.contributor.author Acosta Viana, Karla Y.
dc.contributor.author Brutus, Laurent
dc.contributor.author Ocampo, Susana B.
dc.contributor.author Aznar, Christine
dc.contributor.author Cuba Cuba, Cesar A.
dc.contributor.author Gurtler, Ricardo E.
dc.contributor.author Ramsey, Janine M.
dc.contributor.author Ribeiro, Isabela
dc.contributor.author VandeBerg, John L.
dc.contributor.author Yadon, Zaida E.
dc.contributor.author Osuna, Antonio
dc.contributor.author Schijman, Alejandro G.
dc.date.accessioned 2019-02-06T14:53:10Z
dc.date.available 2019-02-06T14:53:10Z
dc.date.issued 2015
dc.identifier.uri https://hdl.handle.net/20.500.12866/5387
dc.description.abstract BACKGROUND: 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). METHODS/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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. en_US
dc.language.iso eng
dc.publisher PLoS
dc.relation.ispartof urn:issn:1935-2735
dc.rights info:eu-repo/semantics/restrictedAccess
dc.rights.uri https://creativecommons.org/licenses/by-nc-nd/4.0/deed.es
dc.subject Adolescent en_US
dc.subject Adult en_US
dc.subject Female en_US
dc.subject Humans en_US
dc.subject Male en_US
dc.subject Child en_US
dc.subject Child, Preschool en_US
dc.subject Sensitivity and Specificity en_US
dc.subject Genotype en_US
dc.subject Coinfection en_US
dc.subject Genetic Variation/genetics en_US
dc.subject Molecular Typing/methods en_US
dc.subject Biological Assay/methods en_US
dc.subject Chagas Disease/diagnosis/genetics/parasitology en_US
dc.subject Real-Time Polymerase Chain Reaction/methods en_US
dc.subject Trypanosoma cruzi/classification/genetics en_US
dc.title Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples en_US
dc.type info:eu-repo/semantics/article
dc.identifier.doi https://doi.org/10.1371/journal.pntd.0003765
dc.subject.ocde https://purl.org/pe-repo/ocde/ford#3.02.00 es_PE
dc.subject.ocde https://purl.org/pe-repo/ocde/ford#3.03.06

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