Quantitative PCR for Trypanosoma cruzi

Mouse blood samples were mixed with an equal volume of guanidine-HCl 6 M, EDTA 0.1 M, pH 8 (GEB), kept at room temperature for 3 days and then stored at 4°C until use. DNA was isolated from 200 μL of GEB using the High Pure PCR Template preparation Kit (Roche, Mannheim, Germany), according to the manufacturers’ protocol. Parasite DNA was amplified using a T. cruzi satellite DNA sequence of 140 bp flanked by the Sat Fw and Sat Rv oligonucleotides [55 (link)] and SYBR GreenER qPCR SuperMix Universal Kit with integrated uracil DNA glycosylase (UDG; Invitrogen, Life Technologies, Grand Island, NY, USA). DNA amplification was performed in an ABI 7500 thermocycler (Applied Biosystems, Carlsbad, CA, USA) in duplicates using 5 μL of extracted DNA as template (~100 ng) in a final volume of 20 μL. DNA extracted from non-infected mice and samples without DNA template were included as controls. qPCR conditions and standard parasite curve for data analysis were performed as previously described [52 (link),56 (link)].