Abstract
Background Trypanosoma cruzi is the causative agent of Chagas' Disease. The parasite has a complex population structure, with six major evolutionary lineages, some of which have apparently resulted from ancestral hybridization events. Because there are important biological differences between these lineages, strain typing methods are essential to study the T. cruzi species. Currently, there are a number of typing methods available for T. cruzi, each with its own advantages and disadvantages. However, most of these methods are based on the amplification of a variable number of loci.Methodology/Principal FindingsWe present a simple typing assay for T. cruzi, based on the amplification of a single polymorphic locus: the TcSC5D gene. When analyzing sequences from this gene (a putative lathosterol/episterol oxidase) we observed a number of interesting polymorphic sites, including 1 tetra-allelic, and a number of informative tri- and bi-allelic SNPs. Furthermore, some of these SNPs were located within the recognition sequences of two commercially available restriction enzymes. A double digestion with these enzymes generates a unique restriction pattern that allows a simple classification of strains in six major groups, corresponding to DTUs TcI–TcIV, the recently proposed Tcbat lineage, and TcV/TcVI (as a group). Direct sequencing of the amplicon allows the classification of strains into seven groups, including the six currently recognized evolutionary lineages, by analyzing only a few discriminant polymorphic sites.Conclusions/SignificanceBased on these findings we propose a simple typing assay for T. cruzi that requires a single PCR amplification followed either by restriction fragment length polymorphism analysis, or direct sequencing. In the panel of strains tested, the sequencing-based method displays equivalent inter-lineage resolution to recent multi- locus sequence typing assays. Due to their simplicity and low cost, the proposed assays represent a good alternative to rapidly screen strain collections, providing the cornerstone for the development of robust typing strategies.
Highlights
Trypanosoma cruzi, a protozoan parasite of the order Kinetoplastida, is the causative agent of Chagas Disease, a neglected disease that is is endemic in South America, affecting 8 million people in Latin America [1]
We noticed the presence of some tri-allelic sites, and 1 position where all possibilities (A, C, G, and T) were observed in the population. This tetra-allelic SNP alone, at position 657 of the TcSC5D coding sequence, produced a separation of T. cruzi strains in five groups, that matched their classification in discrete typing units (DTUs), as obtained by other methods: DTU I (‘‘T’’ in homozygosity), DTU II (‘‘G’’ in homozygosity), DTU III (‘‘C’’ in homozygosity), DTU IV (‘‘A’’ in homozygosity), and a fifth group consisting of DTUs V and VI (‘‘C’’ and ‘‘G’’ in heterozygosity)
We re-sequenced the TcSC5D gene in an expanded panel of 31 T. cruzi strains from different lineages, confirming the genotype observed for this site in all these strains
Summary
Trypanosoma cruzi, a protozoan parasite of the order Kinetoplastida, is the causative agent of Chagas Disease, a neglected disease that is is endemic in South America, affecting 8 million people in Latin America [1]. A number of protein and genetic markers have been used to classify strains in two [6,7] or three major lineages [6,8,9] Other experimental strategies, such as RAPD and multilocus isoenzyme electrophoresis (MLEE) support the distinction of six subdivisions [10,11,12] referred to as discrete typing units (DTUs), originally designated as DTUs I, IIa, IIb, IIc, IId, and IIe [11]. This nomenclature was revised as follows: TcI, TcII (former TcIIb), TcIII (IIc), TcIV (TcIIa), TcV (TcIId) and TcVI (TcIIe) [13,14], with a seventh DTU (Tcbat/TcVII) proposed around the same time [14,15]. Most of these methods are based on the amplification of a variable number of loci
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