Detecting Plasmodium Species in Blood Samples

DNA was extracted from blood spots on filter papers with the use of InstaGene (Bio-Rad Laboratories, USA) as described previously [40 (link)]. The DNA samples were first examined by nested PCR assays based on the small subunit ribosomal RNA genes of Plasmodium with the aid of genus-specific primers (rPLU1 and rPLU5 in nest 1 amplification, and rPLU3 and rPLU4 in nest 2) as described previously [41 (link)]. Plasmodium-positive samples were then examined by nested PCR assays using species-specific primers to detect P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi, as described previously [42 (link)]. The products of the PCR amplification were analysed by gel electrophoresis in 2.7% agarose gels and were stained by Sybersafe before being observed under UV light.