Fig 1. PCR assay flowsheet¹ to identify Trypanosoma cruzi discrete typing units (DTUs).

Directional arrows indicate assay order and stop signs denote when sufficient data was gathered to theoretically identify the DTU. The final assay (GPI) is included as a confirmatory step, but is not required for DTU identification. ¹Modified from Lewis et al. [37] ²The large subunit rDNA assay is also referred to as the 24sα rRNA gene assay. ³An additional band of approximately 125bp may or may not be visible in combination with the 110bp band. ⁴Heat Shock Protein-60 (HSP60) results in an amplicon of 432-462bp, which upon RFLP with EcoV restriction enzyme yields the following patterns: 1 band (432–462), 2 bands (118–148 + 314), or 3 bands (118–148 + 314 + 432–462). ⁵This PCR used a pool of three primers to amplify a portion of the non-transcribed intergenic region of the tandemly repeated mini-exon gene. ⁶Glucose Phosphate Isomerase (GPI) results in an amplicon of approximately 1264bp, which upon RFLP with HhaI restriction enzyme yields the following patterns: 2 bands (447 + 817), 3 bands (253 + 447 + 490), or 4 bands (253 + 447 + 490 + 817). TcIV will display 2 or 3 bands for North American and South American strains, respectively.