Meltdoctortm hrm master mix

The HRM analyses were performed on an Applied Biosystems 7500 Fast (Life Technologies). All the assays were carried out in duplicate, in a uniplex reaction (containing DNA from a single species of a protozoan), a duplex reaction (containing DNA from two species of protozoa in each reaction: T. gondii + N. caninum, T. gondii + S. neurona, T. gondii + C. parvum, N. caninum + S. neurona, N. caninum + C. parvum, or C. parvum + S. neurona), or a multiplex reaction (containing DNA of the four parasites in a single reaction). For these assays, we used the MeltDoctor^TM HRM Master Mix Kit (Applied Biosystems, USA) in a final volume of 20 μL containing 1× MeltDoctor^TM HRM Master Mix, 0.3 μM each primer, and 40 ng of genomic DNA (or 40 ng of the purified PCR product). The amplification reactions were conducted under the following conditions: initial denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. A melting curve at 95°C for 20 seconds, 95°C for 3 seconds, and 60°C for 30 seconds was recorded after each reaction. The reactions were analyzed by High Resolution Melting (HRM) Software v3.01 (Thermo Fisher Scientific), assessing differences in melting curve shapes and generating three types of graphs represented by derivative, normalized, and difference curves.

Real-time PCR with HRM analysis was performed in a total volume of 10 μL using a 7500 Fast Real-time PCR thermal cycler (Applied Biosystems). The reactions contained 5 μL of MeltDoctor^TM HRM Master Mix (Applied Biosystems), 0.6 μL of each forward and reverse primer, PCR additives, 1 μL of DNA and topped up to 10 μL with double-distilled water. The thermal cycling conditions involved enzyme activation at 95 °C for 10 minutes, followed by 40 cycles of denaturation at 95 °C for 15 seconds and 1 minute of annealing/extending at 60 °C. For the melt curve analysis, the thermal cycling conditions started with denaturation at 95 °C for 10 seconds, annealing at 60 °C for 1 minute, HRM at 95 °C for 15 seconds and finally annealing at 60 °C for 15 seconds. DNA amplification was checked using Applied Biosystems 7500 Software version 2.0.6. The melt curve analysis was performed using the Applied Biosystems HRM Software version 2.0.1.

From each sample, 500 ng genomic DNA was subjected to sodium bisulfite modification using the EZ DNA Methylation-Gold™ Kit (Zymo Research, Irvine, CA, USA) and eluted in a final volume of 110 μl H₂O according to the manufacturer's instructions. Methylation assessment of Region 1, 2 and 3 of the miR-210 promoter was performed by MS-HRM analysis and the primers were designed to amplify both methylated and unmethylated DNA [60 (link)–62 (link)]. PCR and HRM were carried out on a LightCycler^® 480 (Roche, Mannheim, Germany) with each 10 μl reaction comprising 2 × MeltDoctor^TM HRM Master Mix (Life Technologies, Carlsbad, CA, USA), 3 mM MgCl₂, approximately 10 ng bisulfite modified DNA and 500 nM of each primer. The methylation assessment of each region was performed by melting profile comparison between each sample and DNA methylation standards derived through a serial dilution of 100% methylated DNA (Universal Methylated Human DNA Standard, Zymo Research, Irvine, CA USA) into 0% methylated DNA (EpiTect Control DNA, Qiagen, Hilden, Germany). All analyses were done in triplicates and the technical specifications for the Region 1, 2 and 3 MS-HRM assays, including primer sequences, genomic location, assay-specific PCR cycling and HRM protocols are listed in Supplementary Methods.