Iq real time pcr system

Extractions of DNA from vectors and animal tissues were performed using a High Pure PCR Template Preparation Kit (Roche) according to the manufacturer´s instructions. The total DNA was used as a template for Q-PCR amplification with the primers described by Mary et al. [34 (link)] in Bio-Rad iCycler and iQ Real-Time PCR Systems using the SYBR Green detection method (iQ SYBR Green Supermix, Bio-Rad).

Parasite quantification by quantitative PCR (qPCR) was performed in a Bio-Rad iCycler & iQ Real-Time PCR Systems using the SYBR Green detection method (SsoAdvanced Universal SYBR Green Supermix, Bio-Rad, Hercules, CA). Primers targeting the 226 bp long 18S sequence (forward primer 18SN2F 5′-AGA TTA TGG AGC TGT GCG ACA A-3′ and reverse primer 18SN2R 5′-TAG TTC GTC TTG GTG CGG TC-3′) were used. One microlitre of DNA was used per individual reaction. PCR amplifications were performed in duplicates using the following conditions: 98°C for 2:30 min followed by 40 repetitive cycles: 98°C for 10 s and 60°C for 20 s. PCR water was used as a negative control. Detection limit of used assay is 10³ parasites per sample. A series of 10-fold dilutions of L. martiniquensis promastigote DNA, ranging from 5 × 10⁶ to 5 × 10¹ parasites per PCR reaction, was used to prepare a standard curve. Quantitative results were expressed by interpolation with a standard curve. To monitor non-specific products or primer dimers, a melting analysis was performed from 70 to 95°C at the end of each run, with a slope of 0.5°C/c, and 6 s at each temperature. The parasite loads in tested tissues were classified into three categories: low, < 1000 parasites; moderate, 1000–10 000 parasites; heavy, > 10 000 parasites.

On day 8 PI the numbers of Leishmania parasites in individual females were counted using Q-PCR as described previously [31 (link),32 (link)]. Briefly, experimental females were stored at −20°C and total DNA extraction was performed with a High Pure PCR Template Preparation Kit (Roche) according to the manufacturer’s instructions. Kinetoplast DNA was chosen as the molecular target with previously described specific primers (forward: 5´-CTTTTCTGGTCCTCCGGGTAGG-3´; reverse: 5´-CCACCCGGCCCTATTTTACACCAA-3) [32 (link)]. Q-PCR was performed by the SYBR Green detection method (iQSYBER Green Supermix, Bio-Rad, Hercules, CA) in Bio-Rad iCycler & iQ Real-Time PCR systems. Statistical evaluation was performed by the Kruskal-Wallis test using STATISTICA 12 software.