Malaria Journal (May 2017)

Multiplex, DNase-free one-step reverse transcription PCR for Plasmodium 18S rRNA and spliced gametocyte-specific mRNAs

  • Amelia E. Hanron,
  • Zachary P. Billman,
  • Annette M. Seilie,
  • Tayla M. Olsen,
  • Matthew Fishbaugher,
  • Ming Chang,
  • Thomas Rueckle,
  • Nicole Andenmatten,
  • Bryan Greenhouse,
  • Emmanuel Arinaitwe,
  • John Rek,
  • Smita Das,
  • Gonzalo J. Domingo,
  • Kelly Shipman,
  • Stefan H. Kappe,
  • James G. Kublin,
  • Sean C. Murphy

DOI
https://doi.org/10.1186/s12936-017-1863-3
Journal volume & issue
Vol. 16, no. 1
pp. 1 – 13

Abstract

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Abstract Background Plasmodium gametocytes are sexual stages transmitted to female Anopheles mosquitoes. While Plasmodium parasites can be differentiated microscopically on Giemsa-stained blood smears, molecular methods are increasingly used because of their increased sensitivity. Molecular detection of gametocytes requires methods that discriminate between asexual and sexual stage parasites. Commonly tested gametocyte-specific mRNAs are pfs25 and pfs230 detected by reverse transcription polymerase chain reaction (RT-PCR). However, detection of these unspliced mRNA targets requires preceding DNase treatment of nucleic acids to eliminate co-purified genomic DNA. If gametocyte-specific, spliced mRNAs could be identified, DNase treatment could be eliminated and one-step multiplexed molecular methods utilized. Results Expression data was used to identify highly-expressed mRNAs in mature gametocytes that were also low in antisense RNA expression in non-gametocyte stages. After testing numerous candidate mRNAs, the spliced female Pf3D7_0630000 mRNA was selected as a Plasmodium falciparum gametocyte-specific biomarker compatible with Plasmodium 18S rRNA RT-PCR. This mRNA was only detected in samples containing mature gametocytes and was absent in those containing only asexual stage parasites or uninfected human blood. PF3D7_0630000 RT-PCR detected gametocytes across a wide range of parasite densities in both spiked and clinical samples and agreed with pfs25 RT-PCR, the gold standard for RT-PCR-based gametocyte detection. PF3D7_0630000 multiplexed with Plasmodium 18S rRNA RT-PCR was more sensitive than other spliced mRNA targets for one-step RT-PCR gametocyte detection. Conclusions Because the spliced target does not require DNase treatment, the PF3D7_0630000 assay can be multiplexed with Plasmodium 18S rRNA for direct one-step detection of gametocytes from whole human blood.

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