Identification of Trypanosoma cruzi Discrete Typing Units (DTUs) through the implementation of a High-Resolution Melting (HRM) genotyping assay

Abstract

BackgroundChagas 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.FindingsAmplification 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.ConclusionsThe 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.

Identification of Six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay

“…High Resolution Melting (HRM) is a technique that has been widely used in the genotyping of bacteria, fungi, protozoan parasites and vertebrates [25][26][27][28][29] and could be used for identification and genotyping of these Leishmania species [30]. The objective of this study was to evaluate the potential of the HSP70 and ITS1 genes in genotyping six Leishmania species present in the New World through the use of High Resolution Melting (HRM).…”

“…Promastigotes of the reference strains L. panamensis HSP70 and ITS1 genes were used given their good performance as reported by other authors [24][25][26]. For the HSP70 gene, primer design was carried out using the sequences available on GenBank, TriTrypDB and…”

“…[33] since this is a closed-tube assay (does not require additional precautions to prevent crossover of PCR products), avoids sequencing processes, laborious procedures and high costs as well as offering robust and high efficiency in its results [25,34]. On average, this type of test has been calculated to be three times faster and five times cheaper than other types of analysis such as MLST and RFLPs [17].…”

Differential diagnosis of the honey bee trypanosomatids Crithidia mellificae and Lotmaria passim

“…Several loci, including cytochrome b and gGAPDH, showed enough interspecific identity to discriminate between both species, but sequencing remains necessary. Techniques like fragment length polymorphism and high resolution melting (HRM) were previously used to identify trypanosomatids in other host species (Higuera et al, 2013;Schmid-Hempel and Tognazzo, 2010;Zackay et al, 2013). We investigated both methods using infected honey bee samples from different geographic locations and trypanosomatid cell types from honey bees and bumble bees.…”

Rapid detection and identification of four major Schistosoma species by high-resolution melt (HRM) analysis

“…This method greatly simplifies the operation and reduces the analysis time, making it suitable for broad application. In the area of parasitology, it has been adapted recently for the genotyping of apicomplexan parasites (Al-Mohammed 2011;Costa et al 2011;Ngui et al 2012;Zhang et al 2012;Higuera et al 2013;Salim et al 2013). …”