Duplex qPCR for Plasmodium Species Identification

DNA was extracted at NIMPE from 5 mm punches of DBS using QIAamp DNA 96 Blood kit (Qiagen), following manufacturer’s instructions and a final elution in 200 μl of water. A duplex quantitative real-time polymerase chain reaction (qPCR) targeting 18S ribosomal gene of P. falciparum and P. vivax was conducted to confirm species identified by LM [13 (link)]. Briefly, a 7.5 µl mix was prepared using primers and probes from QuantiNova Probe RT-PCR Kit (Qiagen) in a 7500 Real-Time PCR System (Applied Biosystems). Ct value > 40 was considered negative. Samples with no amplification in the Pf–Pv specific qPCR were checked for presence of other Plasmodium species using the generic QMAL qPCR assay [14 (link)].