MULTIPLEX TAQMAN REAL TIME PCR ALGORITHM FOR IDENTIFICATION OF TRYPANOSOMA CRUZI DISCRETE TYPING UNITS

CURA C; DUFFY T; MARCET P.L.; VELAZQUEZ E.; SOSA ESTANI S.; DA SILVA A.; SCHIJMAN A.G

Mar del Plata

Congreso; IX Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2011

Sociedad Argentina de Protozoología

Diversity of Trypanosoma cruzi was extensively demonstrated using different biological, biochemical and molecular strategies targeting several genetic markers, allowing identification of six discrete typing units (DTUs) designated as Tc I – Tc VI (Zingales et al., 2009). In this presentation, we propose a novel algorithm for identifying DTUs, based on two consecutive multiplex real time PCRs using DTUs´specific TaqMan probes. The first PCR strategy involves one common forward primer and four differential reverse primers for amplification of the intergenic region of the spliced leader (SL) genes, and specific LNA Taqman probes for detection of four groups of DTUs: Tc I, Tc III, Tc IV and Tc II/V/VI. Its analytical sensitivity was assessed using purified DNA from reference strains and ranged between 0.5 fg to 1 pg/well depending on which DTU was being detected. The analytical specificity was tested using 27 T. cruzi reference strains from Argentina, Chile, Brazil, Colombia, Mexico and USA, belonging to the six DTUs. SL-multiplex PCR was used to characterize T. cruzi abdomen samples of sylvatic triatomines, isolates from blood cultures of opossums and raccoons from Southern USA, and clinical samples, allowing DTU identification in 39/49 samples, 4 of them being characterized as mixed infections composed by Tc I and Tc IV. The second PCR further discriminates among Tc II, Tc V and Tc VI by amplifying 18S rRNA and cytochrome oxidase subunit II (COII) genes using 3 differential TaqMan probes. This strategy, currently under validation, was found specific to detect DTUs in reference strains. The algorithm herein described, may constitute a methodological improvement in DTU identification, increasing specificity and reducing costs due to the incorporation of TaqMan probes in multiplex reactions, when compared to conventional PCR assays in which a battery of reactions is needed (Burgos et al., Int J Parasitol. 2007).