INVESTIGADORES
FRACCAROLI Laura Virginia
congresos y reuniones científicas
Título:
Adapting a molecular isothermal amplification reaction to develop a simplified Trypanosoma cruzi detection method
Autor/es:
L. LAROCCA; F. STOLOWICZ; L. FRACCAROLI; S. WERBAJH; D. RUIZ; A. VOJNOV; C. CARRILLO
Reunión:
Congreso; XXIX Reunión de la Sociedad Argentina de Protozoología; 2017
Resumen:
Chagas disease, originally from Latin America, is caused by Trypanosoma cruzi. It is estimated that 15000 babies are born withcongenital Chagas around the world each year. Early detectionand treatment of congenital Chagas disease increases the therapy ́s effectiveness. However, serological methods of diagnosis are effective only 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 testing: 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.). The read-out methods were simplified, showing a changing color on 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 as well as for other infectious diseases whose current diagnosis methods also need improvement and simplification.