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Plasmodium species identification and msp-1/msp-2 genotyping

FR Fanomezantsoa Ralinoro
TR Tovonahary Angelo Rakotomanga
RR Rianasoambolanoro Rakotosaona
DR Danielle A. Doll Rakoto
DM Didier Menard
VJ Victor Jeannoda
AR Arsene Ratsimbasoa
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Genus- and species-specific nested PCRs targeting the 18 S rRNA gene were performed, as described by Snounou et al. [10, 17]. The polymorphic regions of the merozoite surface protein genes msp-1 (block 2), msp-2 (block 3) were amplified by nested PCR. In the first round of PCR, oligonucleotide primers were used to target conserved genomic regions within msp-1 (block 2) msp-2 (block 3). In the second round of PCR, the polymorphic families of msp-1 (K1, MAD20, and RO33) and msp-2 (FC27 and 3D7) alleles were amplified with specific primers. The primers and conditions used for first and second rounds of PCR were as described by Oyebola et al. [18]. The PCR products were separated by electrophoresis on a 2% agarose gel, with visualization of the fragments under a gel imager (Gel Doc XR, Biorad) after ethidium bromide staining. The sizes of the alleles (± 20 bp) were determined with molecular weight standards (100 bp DNA Ladder, Invitrogen). DNA from reference P. falciparum strains (3D7, Dd2 and 7G8) was included in each run as a control.

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