Simplified Pan-species Real-time PCR-based Detection of Plasmodium Spp. in Blood Smear

<p><strong><em>Background:</em></strong> We aimed to quicken and simplify the detection of <em>Plasmodium</em> in blood samples by developing and testing a pan-<em>Plasmodium</em> real-time PCR for accurate screening of individuals suspected of malaria.</p><p><strong><em>Methods</em></strong>: A single primer/probe set for pan-species <em>Plasmodium</em>-specific real time PCR targeting a conserved region of the small subunit 18S ribosomal DNA was designed and evaluated for rapid diagnosis and screening of malaria infections using dried blood smears. FTA cards were used for rapid and simple DNA extraction.</p><p><strong><em>Results</em></strong>: The primers and probes showed a positive response with the DNA extracted from bloods infected with<em> P. falciparum</em> and <em>P. vivax</em> but not with DNA extracted from various smears from uninfected blood samples. Seven positive cases positive by both microscopy and nested PCR were found among 280 blood samples taken from in South and Southeast Iran. Five samples were identified as positive for <em>P. vivax</em> and two as<em> </em>positive for <em>P. falciparum</em>. All positive samples were positive by real-time PCR. Furthermore, all 38-blood samples positive by microscopy were positive by real-time PCR. No microscopy-negative samples were positive by real-time PCR.</p><strong><em>Conclusion</em></strong>: By using a simple FTA card for DNA extraction and by application of the real-time PCR developed in this study, sensitivity similar to nested-PCR and microscopy was achieved. This format simplifies the detection of <em>Plasmodium</em> in large numbers of samples