Pcr ready premix

Genomic DNA was extracted from pellets of the Leishmania cultures using High Pure PCR Template Preparation Kit (Roche Diagnostics, Germany) according to the manufacturer′s instructions. Samples were stored at −20 °C until use. DNA samples from Iranian reference strains of L. tropica (MHOM/IR/02/Mash10/Accession No. EF653267), L. major (MRHO/IR/11/GOL-2/ Accession No. JN860745) and L. infantum (MCAN/IR/ 07/Mash-ir1/ Accession No. EU810776) were used as positive controls. The DNA samples were assessed for the Leishmania-specific ribosomal internal transcribed spacer 1 region (ITS1) by PCR amplification using primer pairs of LITSR (F: 5′-CTGGATCATTTTCCGATG-3′) and L5.8S (R: 5′-TGATACCACTTAT CGCACTT-3′). Amplification was carried out using PCR-Ready Premix (Roche, Germany) in a 25μl reaction. The amplification conditions included those described previously (20 (link)). PCR products (8μl) were digested with the restriction endonuclease enzyme HaeIII (BsuRI) (Fermentas, Germany) for the species identification according to the manufacturer′s instructions. Amplicons of nearly 300–350bp and restriction fragments were analyzed using 1.5–3% agarose gels containing safe stain, visualized under UV and compared with those from reference strains of L. tropica, L. major and L. infantum.

CSP gene was amplified using primer pairs of VCS1F 5′-ATG TAG ATC TGT CCA AGG CCA TAA A and VCS1R 5′-TAA TTG AAT AAT GCT AGG ACT AAC AAT ATG (Fig. 1).
Amplification was carried out using PCR-Ready Premix (Roche, Germany) in a 20-μl reaction include 5μL of template DNA, 1 μL of each primer, 10 μL of master mix (amplicon) content 0.4 unit tag polymerase and 0.125mM dntp with 0.8 mM Mgcl2 and 3 μL of distillated water. Reactions were as follow: 95 °C for 5 min, 30 cycle 60 °C for 1 min, 72 °C for 2 min, 94 °C for 1 min followed by 58 °C for 2 min and 72 °C for 5 min, respectively (9 (link)). Then, the product of PCR were analyzed in 1% agarose gels containing 2 μL of simple safe (EURx, Poland) and was visualized under UV. To determine genotypes of VK210, VK247 Using 1100 bp genome fragment the products were then purified using the Nucleo-Spin® Gel and PCR Clean-up purification kit according to manufacture instructions and were send to Bioneer Corporation in South Korea for sequencing.

ITS1 was amplified using specific primers; LITSR (forward: 5′-CTGGATCATTTTCCGATG-3′) and L5.8S (reverse: 5′-TGATACCACTTATCGCACTT-3′). These sets of primers amplify a fragment between 300–360 bp of Leishmania genome [28 (link),29 ]. The post-PCR DNA products were visualized by a UV transluminator, following effective staining with ethidium bromide (safe stain) in 1.2% agarose gel electrophoresis. PCR-Ready premix (Roche, Germany) in 25 μl total reactions comprising 10 μl premix, 2 μl forward and reverse primers (10 pmol), 1 μl DNA template and 13 μl double distilled water was utilized for amplification. Iranian reference strains of L. major (Acc. No: JN860745) and L. tropica (Acc. No: EF653267) were used as positive standard controls in monitoring the reactions. RFLP analysis was deployed in identifying Leishmania species. Purposefully, 10 μl of the PCR product were added to 2 μl of the enzyme buffer, 1 μl of the Fast Digest HaeIII (BsuRI) Enzyme (Fermentas, Life Sciences, Germany) and 17 μl double distilled water. The mixture was incubated at 37°C for 5–10 minutes, as recommended by the manufacturer’s protocol. Separation of the digestion products was discerned by the use of 3% agarose gels, and visualized after effective ethidium bromide staining.