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.
Keywords
Cytochrome b, malaria parasite, plasmodium malariae, mitochondria, real-time PCR.
References
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