Adapting a molecular isothermal amplification reaction to develop a simplified Trypanosoma cruzi detection method

L. LAROCCA; STOLOWICS FG; L. FRACCAROLI,; WERBAJH S; RUIZ D; VOJNOV A; CAROLINA CARRILLO

Congreso; XXIX Reunión de la Sociedad Argentina de Protozoología, en el marco de la Reunión Conjunta de Sociedades de Biociencias; 2017

Chagas disease, originally from Latin America, is caused by Trypanosoma cruzi. It is estimated that 15000 babies/year are born with congenital Chagas around the world. Early-detection and -treatment of congenital Chagas disease increases the therapy´s effectiveness. However, serological methods of diagnostic are effective after 9 months of age, when maternal antibodies have been completely removed; on the other hand, molecular diagnostic strategies, applicable for newborns, require infrastructure, skilled personnel and expensive equipment, factors that restrict their use to few health centers.The objective of this work was to develop a molecular amplification technique to detect T. cruzi DNA with high sensitivity and specificity that would be simple and stable enough to be used on point of care (?POC?) conditions. The first step consisted in selecting target sequences and primer design, using specific bioinformatics software. Then, the in vitro reaction was set up: a- the reaction conditions; b- sensitivity with different types of samples; and c- specificity, using different strains of T. cruzi, other phylogenetically related parasites, human cells and cells unrelated to the parasite or host. Finally, the reaction was adapted to make it simpler and suitable for all health care conditions (different equipment, infrastructure, etc.); particularly, the read out methods were simplified to a changing color and to a lateral flow dipstick (LFD) system.Two of the 6 sets of designed primers, from repetitive DNA fragments, showed high specificity (with no cross reactions) and high sensitivity (~1fg of template), along different types of templates (DNA, parasites, artificially inoculated samples, etc.) and using analytical read out (gel electrophoresis), color changes and LFD.These results encourage us to continue developing a test for Chagas disease and for other infectious diseases whose current diagnosis methods also need improvement and simplification.