Piko real 96 thermocycler

RNA was extracted from aerial tissue collected from five individuals of three new independent experiments with the Direct-zol RNA miniprep kit (Zymo Research, R2050) and digested with the included DNAse I in a column. Total RNA (2.1 μg) was used to synthesize cDNA using the Maxima First Strand cDNA kit (ThermoScientific, K1642). Quantitative PCR was performed in a Piko Real 96 thermocycler (ThermoScientific) using SYBR Green qPCR Master Mix 2× (ThermoScientific, K0251) and 1 μL of 1:10 cDNA dilution (20 μL final volume). Primers were designed using on-line tools at www.idt.com (IDT), with sequences downloaded from phytozome.jgi.doe.gov and synthesized by Macrogen. The efficiency was determined in 1:4, 1:6: 1:64 and 1:256 cDNA dilutions using the integrated thermocycler software (Supplementary Table S6).

The hybrids isolates were evaluated via HRM analyses to observe possible
discriminatory patterns capable of indicating allelic variations. Real-time PCR
assays were performed as described by Zampieri
et al. (2016) in PikoReal96 thermocycler (Thermo Fisher Scientific,
Waltham, MA, USA) using MeltDoctor HRM Master Mix (Life Technologies; Carlsbad,
California, USA) with 50 ng of genomic DNA as the template and the
hsp70F2 (5’-GGAGAACTACGCGTACTCGATGAAG-3’) and
hsp70c reverse primers at 200 nM, as described above. The
cycling conditions were 94 °C for 5 min, followed by 40 cycles of 94 °C for 30 s
and annealing/extension at 60 °C for 30 s, with fluorescent signal acquisition
at the end of each extension step, followed by the dissociation curve for HRM
analysis. The amplicon dissociation analyses were performed via the acquisition
of fluorescence signals at 0.2 °C intervals with holding for 10 s between 60 °C
and 95 °C. Genomic DNA samples from the reference strains L. (L.)
infantum chagasi (MCER/BR/1981/M6445), L. (L.)
amazonensis (MHOM/BR/1973/M2269), L. (L.) mexicana(MNYC/BZ/62/M379), L. (L.) lainsoni (MHOM/BR/81/M6426),
L. (V.) braziliensis (MHOM/BR/1975/M2903), L. (V.)
guyanensis (MHOM/BR/1975/M4147), L. (V.) naiffi(MDAS/BR/1979/M5533) and L. (V.) shawi (MCEB/BR/84/M8408) were
used as standards, and the HRM profiles generated for these species were the
benchmark of the analysis.