Enhanced Sensitivity for Detection of Plasmodium falciparum gametocytes by magnetic nanoparticles combined with enzyme substrate system.

The highly sensitive and specific detection of Pfg377 gene of Plasmodium falciparum gametocyte using Magnetic Nanoparticles PCR Enzyme-Linked Gene Assay (MELGA) was successfully developed. The MELGA included amplification of the Pfg377 gene by polymerase chain reaction (PCR) using magnetic nanoparticles (MNPs)-conjugated forward primer and biotinylated reverse primer, followed by post-analytical process using horseradish peroxidase (HRP)-conjugated streptavidin (SA). The complexes of MELGA product were incubated with the peroxidase substrate and hydrogen peroxide to produce the signal for colorimetric measurement. Altogether, the MELGA technique provided a highly sensitive and specific detection at 1 P. falciparum gametocyte/µL, which was more efficient than that of microscopic examination and rapid diagnostic tests (RDTs). Additionally, the MELGA could detect target gene at femtogram level, which was greater sensitive than the conventional PCR, nested PCR and loop-mediated isothermal amplification (LAMP). The MELGA technique could become a novel and practical method that overcome limitation of traditional gametocyte detection.