Multiplex Taqman Real Time PCR algorithm for identification of Trypanosoma cruzi discrete typing units

DUFFY T.; CURA C.; MARCET P.L.; DA SILVA A.; SCHIJMAN A.G.

Foz do Iguaçu

Congreso; XXVI Reunião Anual da Sociedade Brazileira de Protozoologia. XXXVII Reunião Anual de Pesquisa Básica em Doença de Chagas.; 2010

Sociedade Brazileira de Protozoologia.

MULTIPLEX TAQMAN REAL TIME PCR ALGORITHM FOR IDENTIFICATION OF TRYPANOSOMA CRUZI DISCRETE TYPING UNITS   Duffy, T. 1, Cura, C.I. 1, Marcet P.L. 2, da Silva A. 2, Schijman, A.G. 1* 1LabMECh Dr. Mariano Levin, INGEBI-CONICET, Bs. As., Argentina. 2Centers for Disease Control and Prevention (CDC), Atlanta, USA. *schijman@dna.uba.ar   Diversity of Trypanosoma cruzi was extensively demonstrated using different biological, biochemical and molecular strategies targeting different 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. This strategy 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 isolates from faeces of sylvatic triatomines (Triatoma gerstaeckeri, T. protacta, T. indictiva, T. sanguisuga and T. lecticularia), and from isolates from blood cultures of opossums and raccoons from Southern USA, allowing DTU identification in 31/39 samples, 4 of them being characterized as mixed infections composed by Tc I and Tc IV DTUs. The second PCR further discriminates among Tc II, Tc V and Tc VI DTUs by amplifying 18s rRNA and cytochrome oxidase subunit II (COII) genes using 3 differential TaqMan probes. It was tested using 6 T. cruzi reference strains. The algorithm herein described, currently under standardization, may constitute a methodological improvement in DTU identification, increasing specificity and reducing costs due to the incorporation of TaqMan probes in multiplex reactions and enabling detection of mixed DTU infections, which have been difficult to be detected by conventional PCR assays. Supported by PICT 33955; PIP 2008, CONICET, Argentina.