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

Fecha de publicación:

Autores de IIS La Fe

Participantes ajenos a IIS La Fe

  • Cura CI
  • Duffy T
  • Lucero RH
  • Bisio M
  • Péneau J
  • Jimenez-Coello M
  • Valencia Ayala E
  • Kjos SA
  • Santalla J
  • Mahaney SM
  • Cayo NM
  • Nagel C
  • Barcán L
  • Málaga Machaca ES
  • Acosta Viana KY
  • Brutus L
  • Ocampo SB
  • Aznar C
  • Cuba Cuba CA
  • Gürtler RE
  • Ramsey JM
  • Ribeiro I
  • VandeBerg JL
  • Yadon ZE
  • Osuna A
  • Schijman AG

Grupos

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 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.

Datos de la publicación

ISSN/ISSNe:
1935-2735, 1935-2735

PLOS NEGLECTED TROPICAL DISEASES  PUBLIC LIBRARY SCIENCE

Tipo:
Article
Páginas:
-
Factor de Impacto:
2,444 SCImago ℠
Cuartil:
Q1 SCImago ℠

Citas Recibidas en Web of Science: 66

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