Plasmodium Identification by Nested PCR

Two percent agarose gel electrophoresis of the PCR products was performed followed by GoldView staining and visualized under UV light using Molecular Imager Gel Doc XR⁺ Imaging System (BioRad, CA, United States). All positive results of Plasmodium-infected cases were confirmed again through direct sequencing of the second round nested-PCR products by the BGI Company (China), using nested primers. Sequencing reads were imported into the EditSeq module of Lasergene-Ver7.1¹ to construct ssrRNA gene sequences of the nested-PCR products, which were then blasted at https://blast.ncbi.nlm.nih.gov/. Partial sequences of 18S ribosomal RNA genes of the Togo isolates were aligned with those of other Plasmodium isolates obtained from blast analysis, using the ClustalW method (EMBL-EBI, Hixton and Cambridge, United Kingdom) of MEGA-Ver7.0² to generate a phylogenetic tree based on the neighbor-joining method (Uwase et al., 2020 (link)).