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

Harvard | Vancouver

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. 2017. Multiplex, DNase-free one-step reverse transcription PCR for Plasmodium 18S rRNA and spliced gametocyte-specific mRNAs. [Online]. Figshare. Available from: - https://doi.org/10.6084/m9.figshare.c.3784184

Export Citation

Sharing

Description

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.

Published in a 3rd party system Date: 19 May 2017
Data capture method:
Mode of data capture
Experiment
Data Creators(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

Files

Data not available from this repository.

Dataset may be available externally from:

Related resources

LSHTM Open Access publications:

Resources

Actions

Edit Item Edit Item (admin only)