Multiplex, DNase-free one-step reverse transcription PCR for Plasmodium 18S rRNA and spliced gametocyte-specific mRNAs
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. 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. 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.
Keywords
Gametocyte, mRNA, Spliced, Antisense, Plasmodium, DNaseItem Type | Dataset |
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Capture method | Experiment |
Date | 19 May 2017 |
Language(s) of written materials | English |
Creator(s) | Hanron, AE, Billman, ZP, Seilie, AM, Olsen, TM, Fishbaugher, M, Chang, M, Rueckle, T, Andenmatten, N, Greenhouse, B, Arinaitwe, E, Rek, J, Das, S, Domingo, GJ, Shipman, K, Kappe, SH, Kublin, JG and Murphy, SC |
LSHTM Faculty/Department | Faculty of Infectious and Tropical Diseases > Dept of Clinical Research |
Participating Institutions | London School of Hygiene & Tropical Medicine, London, United Kingdom |
Funders |
Project Funder Grant Number Funder URI |
Date Deposited | 23 May 2017 09:08 |
Last Modified | 08 Feb 2024 17:49 |
Publisher | Figshare |
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