Optimization of DNA extraction from Trypanosoma cruzi by phenol-chloroformisoamyl technique.

ACTIS EA; CAMPO VERDE ARBOCCO F; SUPERINA ME; JAHN GA

Mar del Plata

Congreso; LXI Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica; 2016

Sociedad Argentina de Investigación Clínica

Trypanosoma cruzi is the etiologic agent of Chagas disease, a vector-borne zoonosis widely distributed in the Americas, present in more than 180 species of wild mammals. Eco-epidemiological studies of T. cruzi in wildlife require high sensitivity to obtain representative results. A major constraint of these studies is the low amount of blood that can be collected from small mammals, which may also have low parasitemia. Real-time PCR (qPCR) is a useful and sensible diagnostic technique. DNA extraction can be done using commercial kits, which although optimizing and increasing the DNA output, also increase the processing cost. On the other hand, obtainment of detectable quantities of parasitic DNA using conventional extraction techniques such as phenol-chloroform-isoamyl may be inefficient when parasitemia is low, such as in the chronic stage of the disease. We optimized the performance of the phenol-chloroform-isoamyl technique based on the co-precipitating capacity of mammalian euchromatin to increase the extraction output of parasitic DNA. We worked with different dilutions of T. cruzi epimastigotes (Dm28c clone), with and without supplemented genomic DNA (obtained from rat liver). We extracted DNA by phenol-chloroform-isoamyl technique and performed qPCR with Taq Brazil polymerase and specific primers (121-122) for the DNA kinetoplast of T. cruzi. The correct molecular weight of the amplicon (330 pb) and additional band (≈500pb) was confirmed by 3% agarose gel electrophoresis. In the genomic DNA-supplemented samples, the take-off was 2-3 times lower than without supplemented DNA, indicating that the extraction efficiency and qPCR sensitivity for T. cruzi DNA detection were improved. The addition of genomic DNA significantly increased the efficiency of the extraction technique without contributing external components to a conventional extraction. It is therefore a valuable low-cost tool for T. cruzi detection in cases with low parasitemia.