TY - JOUR
T1 - Identification of Trypanosoma cruzi Discrete Typing Units (DTUs) through the implementation of a High-Resolution Melting (HRM) genotyping assay
AU - Higuera, Sonia L.
AU - Guhl, Felipe
AU - Ramírez, Juan David
N1 - Funding Information:
We thank IRD and LSHTM for providing the DNA of the reference strains and clones in the framework of the Chagas EpiNet project. Financial support was provided by the Project Chagas EpiNet from The European Union Seventh Framework Programme, contract no.223034.
PY - 2013
Y1 - 2013
N2 - Background: Chagas disease, caused by Trypanosoma cruzi, is a geographically widespread anthropozoonosis that is considered a major public health problem in Latin America. Because this parasite presents high genetic variability, a nomenclature has been adopted to classify the parasite into six discrete typing units (DTUs): TcI, TcII, TcIII, TcIV, TcV, and TcVI, which present different eco-epidemiological, clinical, and geographic associations. Currently, the available genotyping methods present a series of drawbacks that implies the need for developing new methods for characterizing T. cruzi DTU's. The aim of this work was to genotype reference populations from T. cruzi by means of a High-Resolution Melting (HRM) genotyping assay.To genotype the DTUs of 38 strains and 14 reference clones of T. cruzi from diverse sources, real-time PCR (qPCR) was coupled to high-resolution melting (HRM) based on the amplification of two molecular markers - the divergent domain of the 24 sα rRNA gene and the intergenic region of the mini-exon gene. Findings. Amplification of the mini-exon gene allowed the genotyping of three distinct groups: TcI, TcII- TcIV- TcV, and TcIII-TcVI, while amplification of the 24sα gene generated non-overlapping melting temperature ranges for each DTU that were used to identify the groups in the six existing DTUs of Trypanosoma cruzi. Conclusions: The proposed genotyping assay allowed discrimination of the six genetic groups by obtaining specific melting curves for each DTU. The application of this technique is proposed because of its specificity, sensitivity, high performance, and low cost compared with other previously described characterization methods.
AB - Background: Chagas disease, caused by Trypanosoma cruzi, is a geographically widespread anthropozoonosis that is considered a major public health problem in Latin America. Because this parasite presents high genetic variability, a nomenclature has been adopted to classify the parasite into six discrete typing units (DTUs): TcI, TcII, TcIII, TcIV, TcV, and TcVI, which present different eco-epidemiological, clinical, and geographic associations. Currently, the available genotyping methods present a series of drawbacks that implies the need for developing new methods for characterizing T. cruzi DTU's. The aim of this work was to genotype reference populations from T. cruzi by means of a High-Resolution Melting (HRM) genotyping assay.To genotype the DTUs of 38 strains and 14 reference clones of T. cruzi from diverse sources, real-time PCR (qPCR) was coupled to high-resolution melting (HRM) based on the amplification of two molecular markers - the divergent domain of the 24 sα rRNA gene and the intergenic region of the mini-exon gene. Findings. Amplification of the mini-exon gene allowed the genotyping of three distinct groups: TcI, TcII- TcIV- TcV, and TcIII-TcVI, while amplification of the 24sα gene generated non-overlapping melting temperature ranges for each DTU that were used to identify the groups in the six existing DTUs of Trypanosoma cruzi. Conclusions: The proposed genotyping assay allowed discrimination of the six genetic groups by obtaining specific melting curves for each DTU. The application of this technique is proposed because of its specificity, sensitivity, high performance, and low cost compared with other previously described characterization methods.
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U2 - 10.1186/1756-3305-6-112
DO - 10.1186/1756-3305-6-112
M3 - Research Article
C2 - 23602078
AN - SCOPUS:84876960644
SN - 1756-3305
VL - 6
JO - Parasites and Vectors
JF - Parasites and Vectors
IS - 1
M1 - 112
ER -