Detection of Plasmodium falciparum by Nested PCR

The DBS samples corresponding to positive and negative blood smear samples were analysed for P. falciparum parasitaemia. Genomic DNA was extracted from DBS samples by boiling in Chelex-100 ((Bio-Rad, Berkeley California,USA) as described elsewhere [48 (link), 49 (link)]. DNA extracts were stored at − 20 °C until further use. Plasmodium falciparum was detected by nested PCR amplification (Bio-Rad T100™ thermal Cycler, Berkeley California, USA) of the 18 small sub-unit ribosomal RNA (18S rRNA) gene [48 (link)] using specific primers (Inqaba Biotec Pretoria, South Africa). Amplification conditions are shown in Additional file 1. Briefly, amplification was done at a total volume of 25μL which included 12.5μL of One Taq quick load 2X with standard buffer (New England Bio Labs), 0.5μL of the forward and reversed primers, 3μL of the DNA template and the rest filled with DNase free water. The amplification conditions for the primary and nested PCR were; Primary PCR of 25 cycles (initial denaturation-94 ºC for 3 min, denaturation-94 ºC for 30 s, annealing-55 ºC for 1 min, extention-68 ºC for 1 min, final extention-68 ºC for 3 min) and nested PCR of 30 cycles (initial denaturation-94 ºC for 3 min, denaturation-94 ºC for 30 s, annealing-61 ºC for 1 min, extention-68 ºC for 1 min, final extention-68 ºC for 3 min). The amplicon was separated in a 2% agarose gel electrophoresis alongside a 100 bp molecular weight maker and observed using a gel documentation system (Molecular Image® Gel Doc^™ XR + System with Image Lab^™ software, Bio-Rad, Berkeley California, USA). An amplified 205 bp indicated P. falciparum infection (Additional file 2(1)) The Nested PCR assay presented allows the detection and identification of P. falciparum at a lower limit of 1–10 parasites/μL [48 (link)].