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Título: Multiplex real-time PCR assay using taqman probes for the identification of trypanosoma cruzi DTUs in biological and clinical samples
Autor(es): Cura, Carolina I.
Duffy, Tomas
Lucero, Raúl H.
Bisio, Margarita
Péneau, Julie
Coello, Matilde Jimenez
Calabuing, Eva
Gimenez, María J.
Ayala, Edward Valencia
Kjos, Sonia A.
Santalla, José
Mahaney, Susan M.
Cayo, Nelly M.
Nagel, Claudia
Barcán, Laura
Machaca, Edith S. Málaga
Viana, Karla Y. Acosta
Brutus, Laurent
Ocampo, Susana B.
Aznar, Christine
Cuba, César Augusto Cuba
Gürtler, Ricardo E.
Ramsey, Janine M.
Ribeiro, Isabela
VandeBerg, Jhon L.
Yadon, Zaida E.
Osuna, Antonio
Schijaman, Alejandro G.
Assunto: Trypanosoma cruzi
Genotipagem
Chagas, Doença de
Algoritmos genéticos
Reação em cadeia de polimerase
Triatoma
Leishmania
Data de publicação: 19-Mai-2015
Editora: PLoS Org.
Referência: CURA, Carolina et al. Multiplex real-time PCR assay using taqman probes for the identification of trypanosoma cruzi DTUs in biological, and clinicial samples. PLoS Neglected Tropical Diseases (Online), v. 9, n.5, p. 1-18, 19 maio 2015. Disponível em: <http://journals.plos.org/plosntds/article/asset?id=10.1371%2Fjournal.pntd.0003765.PDF>. Acesso em: 12 maio 2016. 10.1371/journal.pntd.0003765.
Resumo: 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.
Licença: Copyright: © 2015 Cura et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Fonte: http://journals.plos.org/plosntds/article/asset?id=10.1371%2Fjournal.pntd.0003765.PDF. Acesso em: 15 abr. 2016.
DOI: 10.1371/journal.pntd.0003765 May 19, 2015
Aparece nas coleções:FMD - Artigos publicados em periódicos
UnB - Professores Eméritos

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