Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Hanron AE

PROVIDER: S-EPMC5438523 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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

Hanron Amelia E AE Billman Zachary P ZP Seilie Annette M AM Olsen Tayla M TM Fishbaugher Matthew M Chang Ming M Rueckle Thomas T Andenmatten Nicole N Greenhouse Bryan B Arinaitwe Emmanuel E Rek John J Das Smita S Domingo Gonzalo J GJ Shipman Kelly K Kappe Stefan H SH Kublin James G JG Murphy Sean C SC

Malaria journal 20170519 1

<h4>Background</h4>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 polymera ...[more]

PMID: 28526032

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Dataset Information

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

Publications

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

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