Development of PCR–RFLP assay for the discrimination of Plasmodium species and variants of P. vivax (VK210, VK247 and P. vivax-like) in Anopheles mosquitoes

The identification of Plasmodium species in Anopheles mosquitoes is an integral component of malaria control programs. We developed a new assay to identify Plasmodium falciparum, Plasmodium malariae, and Plasmodium vivax variants. Specific primers were designed to hybridize to CS gene-specific regions. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used to distinguish the P. vivax variants VK210, VK247, and P. vivax-like. The new PCR–RFLP assay revealed good agreement when compared with a nested PCR using artificially infected Anopheles mosquitoes. This sensitive PCR–RFLP method can be useful when detection of Plasmodium species and P. vivax variants is required and may be employed to improve the understanding of malaria transmission dynamics by Anopheles species.