Molecular imager gel doctm xr system with image labtm software

Species-specific primers (Inqaba Biotec, Pretoria, South Africa) targeting the small subunit 18S rRNA Plasmodium gene were used to amplify and characterize Plasmodium species by nested PCR, as previously described [37 (link)]. The resulting amplicons were separated on a 2% agarose gel stained with ethidium bromide and visualised using a gel documentation system (Molecular Imager® Gel DocTM XR + System with Image LabTM Software, Bio-Rad, Berkeley, California, USA). PCR was repeated for all negative samples. Samples that were negative after two PCRs, samples with mixed infections of P. falciparum and other species, and monoinfections with species other than P. falciparum were removed from the study. Only samples with P. falciparum monoinfection were used for subsequent experiments.

Total fungal DNA extraction was conducted following [4 (link)]. Three different fungal gene regions consisting of translation elongation factor 1α (EF-1α) and the largest subunits of RNA polymerase (RPB1 & RPB2) were amplified (Table S2). All PCR reactions were performed using the GeneAmp PCR system 97,000 PCR (Applied Biosystem, Foster City, CA, USA) following the protocol of Tran et al. 2021 [4 (link)]. Amplicons were separated on a 1.5% (w/v) agarose gel, stained with ethidium bromide for 30 min and then visualized using the Molecular Imager^® Gel Doc^TM XR+ System with Image Lab^TM Software (BIO-RAD, Hercules, CA, USA). PCR products were purified using the E.Z.N.A.^® Cycle-Pure Kit (VWR International, Leuven, Belgium) before sending to LGC Genomics (LGC group, Berlin, Germany) for Sanger sequencing.

Portions of the Pfmdr1 gene comprising codons 86, 184, and 1246 were amplified by nested PCR as previously described [38 (link)], using sequence-specific primers purchased from Inqaba Biotech (Pretoria, South Africa) for 400 randomly selected samples, 100 per study site; randomisation was performed with the aid of a computer-generated list. The amplicons were analyzed by restriction fragment length polymorphism (RFLP) using the restriction endonucleases ApoI, DraI, and BglII, as previously described [38 (link)]. The restrictive fragments were resolved on a 3% agarose gel stained with ethidium bromide and later visualised in a gel documentation system (Molecular Imager® Gel DocTM XR + System with Image LabTM Software, Bio-Rad, Berkeley, California, USA).