Detection of Trypanosoma cruzi and Triatominae in Dissected R. prolixus

Each pool, containing the corresponding of 5 digestive tracts of dissected R. prolixus, were pretreated for 2 h at 56°C with 100 μl lysis buffer containing 10 mM Tris-HCl (pH 9.2), 1 mM EDTA and 150 μg/ml proteinase K (Sigma-Aldrich, St. Louis, MO, USA). DNA was purified from the lysate using QIAamp DNA mini kit (Qiagen, Hilden, Germany) and resuspended from the silica column in a final volume of 100 μL of elution buffer from the kit [11 (link)]. DNA was stored at– 20°C until further analysis.
Multiplex qPCR reactions were carried out in a final volume of 20 μl, containing 2 μl DNA (8–10 ng), 2× FastStart TaqMan Probe Master Mix (Roche applied science, Mannheim, Germany), 600 nM cruzi1/cruzi2 primers and 250 nM Cruzi3 probe (FAM/NFQ-MGB) targeting T. cruzi nuclear satellite DNA (SAT-DNA), 300 nM P2B primer, 500 nM P6R primer and 150 nM Triat Probe (VIC/NFQ-MGB) (Applied Biosystems) targeting the 12S ribosomal subunit gene of triatomines [11 (link),19 (link),22 (link)]. Sequences of both sets of primers and probes are presented in Table 1. The cycling conditions were as follows: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 58°C for 1 min. Amplifications were performed in the ABI Prism 7500 Fast (Applied Biosystems).