Publications des scientifiques de l'IRD

Cura C. I., Duffy T., Lucero R. H., Bisio M., Peneau J., Jimenez-Coello M., Calabuig E., Gimenez M. J., Ayala E. V., Kjos S. A., Santalla J., Mahaney S. M., Cayo N. M., Nagel C., Barcan L., Machaca E. S. M., Viana K. Y. A., Brutus Laurent, Ocampo S. B., Aznar C., Cuba C. A. C., Gurtler R. E., Ramsey J. M., Ribeiro I., VandeBerg J. L., Yadon Z. E., Osuna A., Schijman A. G. (2015). Multiplex real-time PCR assay using TaqMan probes for the identification of Trypanosoma cruzi DTUs in biological and clinical samples. Plos Neglected Tropical Diseases, 9 (5), p. e0003765 [18 p.]. ISSN 1935-2735.

Titre du document
Multiplex real-time PCR assay using TaqMan probes for the identification of Trypanosoma cruzi DTUs in biological and clinical samples
Année de publication
2015
Type de document
Article référencé dans le Web of Science WOS:000355303600032
Auteurs
Cura C. I., Duffy T., Lucero R. H., Bisio M., Peneau J., Jimenez-Coello M., Calabuig E., Gimenez M. J., Ayala E. V., Kjos S. A., Santalla J., Mahaney S. M., Cayo N. M., Nagel C., Barcan L., Machaca E. S. M., Viana K. Y. A., Brutus Laurent, Ocampo S. B., Aznar C., Cuba C. A. C., Gurtler R. E., Ramsey J. M., Ribeiro I., VandeBerg J. L., Yadon Z. E., Osuna A., Schijman A. G.
Source
Plos Neglected Tropical Diseases, 2015, 9 (5), p. e0003765 [18 p.] ISSN 1935-2735
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 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.
Plan de classement
Sciences fondamentales / Techniques d'analyse et de recherche [020] ; Entomologie médicale / Parasitologie / Virologie [052] ; Sciences du monde animal [080]
Description Géographique
AMERIQUE DU SUD ; ETATS UNIS
Localisation
Fonds IRD [F B010064658]
Identifiant IRD
fdi:010064658
Contact