Identification of Trypanosoma cruzi Discrete Typing Units by Multiplex Real-Time PCR.

Trypanosoma cruzi isolates are classified into six discrete typing units (DTUs), TcI to TcVI, and TcBat. An accurate identification of T. cruzi genotype is critical for understanding its epidemiology, pathogenesis, and response to treatment. Several typing methodologies have been developed with variable levels of complexity, selectivity, and analytical sensitivity. This chapter describes a standardized multiplex real-time polymerase chain reaction (PCR) protocol using TaqMan probes to identify parasite DTUs based on the analysis of T. cruzi SL-IR, 18S rDNA, COII, and 24Sα rDNA targets. This methodology can identify the six main DTUs directly from clinical and biological specimens in a single or two sequential qPCR reactions with high sensitivity and specificity, minimizing contamination risks and reducing the number of steps required. It offers a simplified approach to genotyping that is adaptable for field studies and clinical diagnostics.