Detecting Plasmodium vivax in Colombian Samples

Parasitized DNA presence and integrity in 100 samples stored at -20°C (2007-2010) at FIDIC (from different areas of Colombia) were evaluated by 18S ribosomal RNA gene amplification using specific primers for P. vivax (SSU-F 5′-ATGAACGAGATCTTAACCTGC-3′ and SSU-R 5′-CATCACGATATGTA5TGATAAAGATTACC-3′) in a touchdown PCR [41 (link)]. The reaction contained: 1x Mango Taq reaction buffer (Bioline), 2.5 mM MgCl₂, 0.25 mM dNTPs, 0.5 mM of each primer, 0.1 U Mango Taq DNA polymerase (Bioline) and 10-40 ng gDNA in 10 mL final volume. The PCR thermal profile was: one initial denaturing cycle at 95°C (5 min), followed by ten cycles at 95°C (20 sec), annealing at 65°C (30 sec) and an extension step at 72°C (45 sec). Annealing temperature was reduced by 1°C in each cycle until reaching 55°C; 35 additional cycles were run at this temperature followed by a final extension cycle at 72°C (10 min). PCR products were visualized by electrophoresis on 1.5% agarose gel in 1× TAE, using 1 μL SYBR-Safe (Invitrogen).