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

Abstract

Chagas disease, caused by the protozoan Trypanosoma cruzi, represents a health and social threat to an estimated number of eight million people, affecting mainly neglected populations in endemic areas and emerging in non endemic countries by migratory movements. Parasite genetic diversity is related to geographical distribution and transmission cycles and might play a role in clinical manifestations as well as in anti-parasitic chemotherapy response. T. cruzi has been classified into six Discrete Typing Units (DTUs), after consensus reached among experts in the field. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is needed. Available typing schemes are usually applied to cultured parasite stocks, because they are not sensitive enough to be used in biological specimens. Only nested PCR procedures could directly type biological samples, but are prompt to contamination and require a high number of reactions. Thus, we developed a multiplex Real-Time PCR using TaqMan probes (MTq-PCR) for DTU typing in a single or a second round of amplification. It proved useful to determine DTUs in cultured stocks, vector and reservoir specimens, as well as in patients´ samples, especially in those from individuals with acute, congenital infection or Chagas reactivation. It is amenable to quantification and automation for kit production.

Citation

Cura, C.I.; Duffy, T.; Lucero, R.H.; Bisio, M.; Péneau, J.; Jimenez-Coello, M.; Calabuig, E.; Gimenez, M.J.; Valencia Ayala, E.; Kjos, S.A.; Santalla, J.; Mahaney, S.M.; Cayo, N.M.; Nagel, C.; Barcán, L; Málaga Machaca, E.S.; Acosta Viana, K.Y.; Brutus, L.; Ocampo, S.B.; Aznar, C.; Cuba Cuba, C.A.; Gürtler, R.E.; Ramsey, J.M.; Ribeiro, I.; VandeBerg, J.L.; Yadon, Z.E.; Osuna, A.; Schijman, A.G. Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples. PLOS Neglected Tropical Diseases (2015) 9 (5) e0003765. [DOI: 10.1371/journal.pntd.0003765]

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

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