Nested PCR for P. falciparum 18S rRNA

The Nested PCR amplification of the P. falciparum 18S rRNA gene was adapted from Singh et al. [36 (link)] with slight modification as previously reported [12 (link)]. Briefly, 200 nM dNTPs, 2 mM MgCl₂, 133 nM each of forward (rPLU6) and reverse (rPLU5) primers (Additional file 1: Table S1) and 1 U OneTaq DNA polymerase (NEB, UK) was used to amplify the 18S rRNA gene from 5 μL (~ 20 ng) of DNA in the primary PCR. The secondary PCR was performed using similar concentrations of reagents as in the primary reaction mix; however, rFal1 (forward) and rFal2 (reverse) primers were used to amplify 1 μL of the primary product. Genomic DNA from the 3D7 strain of P. falciparum (MRA 102G) was used as the positive control sample and distilled water (no template) served as the negative control sample. Positive and negative control samples were included in each PCR reaction set up. The amplified PCR products were separated alongside a 100 bp ladder (New England Biolabs, UK) on a 2% agarose gel stained with Ethidium bromide. The gels were subsequently viewed under ultra-violet light using the FUSION-FX7 advanced (Vilber Lourmat, Germany) chemiluminescence documentation system. All PCR assays were performed using the Eppendorf Mastercycler Nexus thermal cycler (Eppendorf, UK).