Quantifying Trypanosoma cruzi DNA in Tissues

Organs and tissues were snap frozen on dry ice and stored at −80°C. For DNA extraction, samples were thawed and immediately homogenized in at least 200 μl lysis buffer (4 M urea, 200 mM Tris, 20 mM NaCl, 200 mM EDTA, pH 7.4) per 50 mg tissue using a BulletBlender Storm instrument (Next Advance). The tissue suspension was then incubated overnight at 37°C with 0.6 mg proteinase K. DNA was extracted using the High Pure PCR product purification kit (Roche). Real-time PCR reactions were prepared using the QuantiTect SYBR Green PCR Kit (Qiagen) and run on a RotorGene 3000 instrument. Each reaction contained 50 ng DNA and 0.5 μM of each primer. T. cruzi-specific primers TCZ-F and TCZ-R (Cummings and Tarleton, 2003 (link)), targeted at the 195 bp satellite repeat, and mouse-specific primers GAPDHf and GAPDHr (Cencig et al., 2011 (link)), targeted at the murine gapdh gene, were used. Measurements of T. cruzi DNA content were normalized using the ratio of Ct values for T. cruzi- and mouse-specific PCRs, and converted to estimated numbers of parasite equivalents by reference to a standard curve with a range of 2.5 × 10⁶–2.5 × 10⁻¹ parasite equivalents ml⁻¹. The standard curve was established from serial dilution of a DNA sample derived from 75 mg homogenized muscle tissue, spiked with 2 × 10⁷ epimastigotes, using DNA from unspiked equivalent samples as the diluent.