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This article aims to establish a novel cytochrome b real-time PCR assay using Taqman probe for identification of P. malariae and its discrimination from other Plasmodium human infecting species. The optimization of real-time PCR assay with 1X QuantiTect Probe PCR Master Mix, primers and probe used at concentrations of 0.4 μM and 0.1 μM, respectively; and 2.5 mM MgCl2, 5 μl DNA template and deionized H2O of 20 μl, was performed using a real-time PCR instrument. The developed real-time PCR assay was evaluated for the limit of detection, stability on standard panels (109-100 copies/ µl), as well as the sensitivity, specificity on control groups. The probit analysis demonstrates that the 95% detection limit was <0.5 parasite/μl, both the sensitivity and specificity of the assay were 100% when evaluated on the control groups. Additionally, the assay initially evaluated on 41 clinical samples including 21 malaria samples and 20 samples of volunteer blood donors, identified 1 positive sample with P. malariae from the disease group, which shows a concordant result with nested PCR. This novel Cyt b real-time PCR assay for identifying P. malariae may also facilitate earlier discrimination of P. malariae from other Plasmodium parasites with high sensitivity.
Cytochrome b, malaria parasite, plasmodium malariae, mitochondria, real-time PCR.
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