Multiplex, DNase-free one-step reverse transcription PCR for Plasmodium 18S rRNA and spliced gametocyte-specific mRNAs
- Amelia E. Hanron1,
- Zachary P. Billman1, 3,
- Annette M. Seilie1,
- Tayla M. Olsen1,
- Matthew Fishbaugher4,
- Ming Chang1,
- Thomas Rueckle5,
- Nicole Andenmatten5,
- Bryan Greenhouse6,
- Emmanuel Arinaitwe7, 8,
- John Rek7,
- Smita Das9,
- Gonzalo J. Domingo9,
- Kelly Shipman10,
- Stefan H. Kappe4,
- James G. Kublin10 and
- Sean C. Murphy1, 2, 3, 4Email author
© The Author(s) 2017
Received: 6 October 2016
Accepted: 13 May 2017
Published: 19 May 2017
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.
Plasmodium gametocytes are the male and female sexual stages of the parasite responsible for transmission from an infected host into the female Anopheles mosquito vector. While gametocytes do not directly cause clinical disease in the mammalian host, their presence denotes potential continued transmission. Mature gametocytes circulate in peripheral blood for 3 weeks or longer [1–4]. The frequency and density of gametocyte carriage is correlated with the likelihood that mosquitoes will become infected after taking a blood meal [5–7]. Persons with sub-microscopic Plasmodium falciparum gametocyte densities can contribute to transmission [2, 7–9] with transmission possible at densities as low as 250–300 gametocytes/mL of blood . Thus, gametocyte detection strategies should ideally be able to achieve this level of analytical sensitivity.
Gametocytes can be identified by light microscopy of Giemsa-stained thick and thin blood smears but, like all microscopic methods for Plasmodium parasites, only to a density of ~5000–20,000 parasites/mL (5–20/μL) by thick blood smear . Molecular methods are more sensitive and include qualitative or quantitative RT-PCR [3, 11–14] and NASBA [9, 15, 16]. mRNA-based methods are used to differentiate gametocytes from asexual stages by detecting stage-specific mRNAs. The most common gametocyte mRNA targets are pfs25 [17, 18] and pfs230 [18–20] for P. falciparum and pvs25 for P. vivax . These well-studied targets are all derived from unspliced mRNAs, so a DNase treatment step is required to destroy genomic DNA prior to RT. When manual DNase treatment is performed, there is partial loss of sample material and increased risk for sample cross-contamination due to added handling steps. DNase treatment also makes the process more time consuming. For detection of asexual stage parasites, some laboratories already perform one-step multiplex Plasmodium 18S rRNA RT-PCR directly from extracted whole blood without DNase treatment [21, 22]. In these assays, DNase treatment is not required because Plasmodium 18S rRNAs are more than three orders of magnitude more abundant than the coding 18S rDNA genes [21, 22]. Plasmodium 18S rRNAs are developmentally regulated between sexual and asexual stages [23–25] but gametocytes express both S- (sexual) and A- (asexual)-type 18S rRNAs . Because of this expression, 18S rRNAs alone cannot be used to differentiate gametocytes from asexual stage parasites. Spliced mRNAs that were highly expressed in gametocytes and showed nearly absent antisense RNA expression in the asexual stage were hypothesized to be ideal targets for multiplexing with the Plasmodium 18S rRNA assay for one-step RT-PCR Plasmodium detection. Although three spliced gametocyte-expressed mRNAs have been reported as RT-PCR targets [26, 27], it was unknown whether these targets were suitable for multiplexing with the 18S rRNA assay.
Here, bioinformatic methods were used to search for spliced, gametocyte-specific mRNA targets compatible with one-step Plasmodium 18S rRNA RT-PCR methods. After testing candidate targets and identifying several gametocyte-specific spliced mRNAs, the PF3D7_0630000 mRNA was selected as a P. falciparum-specific spliced mRNA for RT-PCR without DNase treatment. This novel mRNA target was then tested against pfs25 and a known spliced mRNA using clinical samples.
mRNA expression fold-change from Lopez-Barragan et al.  was evaluated. Fold-change data was summed for asexual or gametocyte stages, sorted and filtered to remove mRNAs with total asexual expression ≥0 or total gametocyte expression ≤0 (all relative to ring stage expression). Single exon genes were eliminated as were those lacking data in http://www.plasmodb.org and those representing redundant mRNA isoforms. Fold change data in  were based on RNASeq reads per kilobase of exon per million (RPKM). Since RPKM can originate from sense or antisense transcripts, the strand-specific fragments per kilobase of exon per million (FPKM)  were also inspected, and genes with schizont antisense FPKM equal to 50–100% of maximum were eliminated as were those with minimum asexual antisense FPKM within twofold of the maximum gametocyte antisense FPKM values. Remaining genes were sorted on stage V gametocyte expression, and the most highly expressed genes were evaluated for suitable intron-spanning splice RT-PCR designs. RPKM and FPKM data deposited in plasmodb.org from  for selected genes is in Additional file 1.
Primers and probes were designed to target exons using the web-based PrimerQuest tool (http://www.idtdna.com/Primerquest/). Primers were commercially synthesized (IDT DNA, Coralville, IA, USA); primer set names (S1, S2, S3, etc.) were arbitrarily assigned by the IDT PrimerQuest software.
Plasmodium falciparum cultures
Asexual stage Plasmodium parasites were cultured as described . Gametocytes were cultured as described . Purity of cultures was confirmed by microscopy. Some asexual stage cultures were further confirmed gametocyte-free by pfs25 RT-PCR.
Human clinical samples
Leftover clinical whole blood specimens (50 μL) were preserved in 2 mL of NucliSens Lysis Buffer (bioMérieux, Marcy-l’Étoile, France). These samples were used under a protocol approved by the University of Washington Institutional Review Board (IRB) (protocol 47026, S. Murphy). Samples were included from a clinical trial of a novel chemical entity DSM265 approved under Fred Hutchinson Cancer Research Center IRB (protocol 8408, J. Kublin). Samples from a field study in Uganda were collected from consenting study participants or with the assent of children and consenting legal care givers within the context of studies approved by the relevant IRBs. Specimens from Nagongera, Tororo District, Uganda were collected under a study approved by the University of California San Francisco IRB 11-05995 and Uganda IRB 2011-0167. In Uganda, EDTA whole blood samples were collected from a previously described surveillance cohort ; all participants provided informed consent. Samples from Uganda were collected from May to October 2015.
Nucleic acid extraction
Nucleic acids were extracted on a bioMérieux EasyMAG  or an Abbott m2000sp as described . Briefly, on the bioMérieux instrument, 1 mL of the 2.05 mL lysed sample (50 µL whole blood plus 2 mL NucliSENS lysis buffer) was extracted for total nucleic acids and eluted into 40 µL of elution buffer. On the Abbott m2000sp instrument, 1 mL of the 2.05 mL lysed sample was extracted preferentially for RNA and eluted into 53 μL of elution buffer.
In initial studies of candidate primers, singleplex RT-PCR was performed using the AgPath mastermix kit as described by the manufacturer (Thermo Fisher Scientific, Waltham, MA) in the presence of LCGreen DNA-binding dye as described by the manufacturer (BioFire, Salt Lake City, UT). Some such RT-PCR products were evaluated by 1.5% agarose gel electrophoresis. For multiplex studies of spliced mRNA targets with 18S rRNA, pan-Plasmodium 18S rRNA RT-PCR was performed as described  with the addition of the candidate spliced gametocyte primers (200 nM) and probe (100 nM) on an Abbott m2000rt or Bio-Rad C1000 (Bio-Rad, Hercules, CA). Briefly, 15 µL of eluate was combined with 35 µL of mastermix as described in the SensiFAST LO-ROX one step RT-PCR kit product manual (Bioline, Tauton, MA). Cycling conditions were reverse transcription 10 min at 48 °C, 95 °C for 2 min followed by 45 cycles of 95 °C for 5 s followed by 50 °C for 35 s. For pfs25/18S rRNA multiplex RT-PCR, total nucleic acids were DNase treated (TURBO DNA-free Kit, Ambion/Life) and pfs25 and 18S rRNA RT-PCR performed using published cycling conditions and concentrations of Plasmodium 18S rRNA primers/probe  supplemented with pfs25 primers (400 nM) and probe (200 nM) . Reagents for a gametocyte-specific mRNA from P. falciparum meiotic recombination protein DMC1-like protein gene (PF3D7_0816800) were as previously reported : forward 5′-ATATCGGCAGCGAAAATGTGT-3′; reverse 5′-GACAATTCCCCTCTTCCACTGA-3′; probe 5′-(6-FAM)-TGCCCTTCTCGTAGTTGATTCGATTATT(BHQ1)-3′. Reagents for the early/mid- (PF3D7_1477700/PF14_0748) and mid/late-gametocyte expressed mRNAs (PF3D7_1438800/PF14_0367) were as previously reported : PF3D7_1477700: forward 5′-CTTATGTGCTGAATTTTGTGTTATGGT-3′, reverse 5′-TTGGCCACACTGCTCTAGGA-3′, probe: 5′(VIC)-CACATAATGAATTCAAGGGTAG(MGBNFQ)-3′; PF3D7_1438800: forward 5′- GTTACATTTCGACCCAGCATAAATT-3′, reverse 5′-TCCCTGTGTTTTTGCTCATCTTC-3′, probe 5′-(VIC)-CAGTGCATATTGTTGCCTGT(MGBNFQ)-3′. All probes were 6-FAM-labelled and sourced from IDT (Coralville, IA) with the exceptions of the CAL Fluor orange 560-labeled pan-Plasmodium 18S rRNA hydrolysis probe (LCG Biosearch Technologies, Novato, CA) and the VIC-labeled PF3D7_1477700 and PF3D7_1438800 probes (Invitrogen). Where indicated, some reactions were tested at other annealing temperatures to investigate specificity. Quantification of parasites was achieved using an absolute 18S rRNA standard curve as described [22, 31], which allowed estimation of gametocyte-specific assay limits of detection (LOD). For gametocyte markers evaluated using whole parasite standard curves, CT values up to 40.0 cycles were converted into quantitative values.
To evaluate unlysed sample stability, an unpaired t test was used to compare mean CT values for 18S rRNA and gametocyte-specific markers and the difference in these CT values. For sensitivity analyses, exact confidence intervals were calculated . Statistical significance for both was P < 0.05.
One-thousand one-hundred twenty-nine genes evaluated previously by RNAseq  were filtered to remove those with increased asexual expression (sum of asexual fold change ≥0) and decreased gametocyte expression (sum of stage II and V gametocyte fold change ≤0). This resulted in 372 genes with gametocyte stage II and/or V expression and an absence of asexual-stage expression. The most well-known gametocyte target pfs25 (PF3D7_1031000) was one of the most highly expressed gametocyte-specific genes in this set (Additional file 1). Six genes were eliminated because they lacked data in plasmodb.org and all single exon genes were also eliminated. Two-hundred genes contained two or more exons. Since antisense transcripts are produced for many Plasmodium genes  and since such transcripts can lead to false positive results in primer-specific RT-PCRs [33–36], antisense strand-specific FPKM data  were evaluated next. Genes with low or absent antisense transcript expression in non-gametocyte stages were retained (48 genes), sorted on stage V gametocyte expression and evaluated for intron-spanning primer/probe designs compatible with the Plasmodium 18S rRNA RT-PCR cycling temperatures. RT-PCR designs for 14 genes were ultimately selected using this strategy as were designs for four additional genes overexpressed in stage II/V gametocytes but not identified by published fold-change data in ; published RT-PCRs for pfs25 and three reported multi-exon gametocyte-specific genes [26, 27] were also included (Additional file 1). Based on recently published data , two genes were male-specific (PF3D7_1477700, PF3D7_1438800), one showed mixed expression (PF3D7_1020100) and the rest were female-specific (Additional file 1).
RT-PCR with LCGreen intercalating dye for candidate targets
Candidate primer/probe combinations evaluated for gametocyte-specific targets
Cycle thresholds for Plasmodium 18S rRNA, PF3D7_0630000 and pfs25 in gametocyte dilution series
Plasmodium 18S rRNA
3 × 107
3 × 106
3 × 105
3 × 104
3 × 103
3 × 102
3 × 101
3 × 100
PF3D7_0630000 mRNA stability
Kinetics of PF3D7_0630000 mRNA expression
Detection of gametocytaemia in a volunteer in a controlled human malaria infection trial
Multiplex comparison against other reported gametocyte-specific spliced mRNA targets
Example cycle thresholds for PF3D7_0630000 and published gametocyte-specific RT-PCRs
RT-PCR target (annealing temp.)
Gametocyte 3 × 106 g/mL
4 × 105 p/mL
8 × 102 p/mL
PF3D7_0630000 (50 °C)
PF3D7_0816800 (50 °C)
PF3D7_1477700 (50 °C)
PF3D7_1477700 (60 °C)
PF3D7_1438800 (50 °C)
PF3D7_1438800 (60 °C)
pfs25 (50 °C, DNased template)
Plasmodium 18S rRNA
One-step multiplex detection of gametocytes in asymptomatic subjects in Uganda
Performance of gametocyte-specific RT-PCR markers against field samples from Uganda
Sensitivity (95% CI)
1–100 (n = 20)
101–1000 (n = 13)
>1000 (n = 14)
All samples (n = 74)
Specificity (95% CI)
All samples (=74)
Quantification of gametocytes in field samples based on pfs25 resulted in estimated densities 1.18 log10 parasites/mL (95% CI 0.89–1.46 log10 parasites/mL) lower on average than the paired estimates made with PF3D7_0630000. Lower pfs25-derived gametocyte quantification could not be explained by overall loss of total nucleic acids during DNase treatment since 35/39 evaluable paired samples (positive by pfs25 and PF3D7_0630000) actually showed earlier 18S rRNA CTs in the pfs25 sample compared to the PF3D7_0630000 sample (Additional file 3).
Plasmodium gametocytes can be detected with mRNA-based molecular methods at sub-microscopic densities. Commonly targeted gametocyte-specific P. falciparum mRNAs include those made from single exon genes pfs25 and pfs230 [17–20], which necessitates DNase treatment prior to RT-PCR. Here, based on published RNAseq data , 18 gametocyte-specific, multi-exon mRNAs were identified and evaluated as DNase-free gametocyte targets to determine if they could be multiplexed with an existing highly sensitive Plasmodium 18S rRNA qRT-PCR. The bioinformatics strategy employed here selected for genes that were highly expressed in mature gametocytes and showed near-zero sense or antisense expression in asexual stages. In wet-lab testing, two female gametocyte-specific mRNAs showed suitable multiplex RT-PCR target characteristics: PF3D7_0630000 and PF3D7_0514500. PF3D7_0630000 encodes a CPW-WPC protein likely expressed in ookinetes, though the coding mRNAs are first expressed and post-transcriptionally regulated in gametocytes . Since the PF3D7_0630000 mRNA accumulates in mature female gametocytes as the parasite awaits ookinete formation, this target may be an ideal gametocyte marker in human blood. PF3D7_0514500 (alias PFE0725C) encodes a six-exon conserved membrane protein of unknown function and was noted to be a member of the sexual development gene cluster in a previous full-genome high-density oligonucleotide microarray study . Interestingly, PF3D7_0630000 was absent from the same gene cluster study.
Since PF3D7_0630000 was the most promising target, it was intended to be compared against RT-PCR assays for pfs25 and for three other known spliced gametocyte-expressed mRNAs: PF3D7_0816800, PF3D7_1477700 and PF3D7_1438800. However, two of the intended comparator targets could not be multiplexed with the 18S rRNA RT-PCR assay and were therefore not studied against PF3D7_0630000. These targets produced positive results when tested against microscopically-pure asexual stage cultures (that were also pfs25-negative). The extraction method and RT-PCR mastermix used here differed from that originally reported for these markers , which may account for discrepancies. In addition, unlike the other targets studied in this project, the PF3D7_1477700 and PF3D7_1438800 mRNAs were both male gametocyte-specific , which could have led to false positives in asexual samples if male gametocytes (or less apparent exflagellated forms) were present at minuscule concentrations in asexual cultures; such forms would also be pfs25-negative. While PF3D7_1477700 and PF3D7_1438800 mRNAs may be suitable for multiplexing with 18S rRNA in another extraction/mastermix system, this possibility was not further evaluated here.
The PF3D7_0630000 multiplex RT-PCR was comparably sensitive to pfs25 when tested against a samples from asymptomatic Ugandan subjects. Given this performance, PF3D7_0630000 RT-PCR will be able to detect gametocytes at densities that contribute to transmission, although this should be studied in future in prospective studies. Interestingly, PF3D7_0630000-derived gametocyte density estimates were 1.18 log10 higher on average than pfs25-based estimates, which likely reflects degradation of pfs25 mRNA (but not more robust 18S rRNA) during the DNase treatment step. Detection of pfs25 mRNA by QT-NASBA (which does not require DNase treatment) was reportedly more sensitive than pfs25 RT-PCR, and the authors postulated that the DNase treatment step could have reduced the sensitivity of pfs25 RT-PCR . Thus, it is likely that the increased sensitivity of pfs25 RT-PCR (due to its increased expression) is somewhat offset by increased degradation at the DNase treatment step.
By eliminating DNase treatment, gametocyte-specific RT-PCR targets can be directly incorporated into multiplex RT-PCR assays from total nucleic acids. This importance of this workflow improvement may be significant. Although ‘on-column’ DNase treatment is available for some manual RNA purification kits, this was not an option for the platform used here (Abbott m2000sp/rt . Elimination of DNase treatment would be advantageous. In epidemiological studies, simplified sample processing that minimizes hands-on time and eliminates manual steps are desirable since extra steps serve to increase false positive and negative results through cross-contamination, target degradation and other processing errors. The use of spliced gametocyte-specific mRNAs such as those identified here (PF3D7_0630000 and PF3D7_0514500) or those reported previously [26, 27] offer the possibility of this sort of simplified testing. Other spliced mRNA targets beyond these may also be suitable for RT-PCR. Similarly, spliced mRNA RT-PCR may also be useful for detecting asexual stage-specific spliced mRNAs such as the ring-specific transcript from the two-exon PF3D7_0501300 (PFE0065w) gene previously used as an asexual parasite marker . Multiplex assays that include the Plasmodium 18S rRNA, a spliced gametocyte-specific mRNA and a spliced ring/asexual-specific mRNA could eventually provide for one-step P. falciparum infection monitoring that would differentiate between potentially symptomatic and asymptomatic infections. The PF3D7_0630000-specific spliced marker identified here may be useful in future gametocyte screening studies.
AH and SCM performed the bioinformatics analyses. AH, ZPB, TMO, AMS, MC and SCM developed RT-PCR reagents and performed the experiments. MF and SHK cultured P. falciparum gametocytes. TR, NA, KJ, JGK and SCM performed the DSM265 clinical trial. BG, EA, JK, SD and GJD performed the field study. AH, ZPB and SCM reviewed the data and wrote the manuscript that was reviewed. All authors read and approved the final manuscript.
The staff of the Insectary at the Center for Infectious Disease Research is acknowledged for assistance with parasite culture. Staff at the Fred Hutchinson Cancer Research Center Prevention Center, the Seattle Malaria Clinical Trials Center and the Medicines for Malaria Venture are acknowledged for assistance with the DSM265 trial. Staff at the clinical site in Uganda are acknowledged for conducting the field study.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets utilized in this manuscript are described in the manuscript or are available at plasmodb.org and in Ref. .
Consent to publish
The IRB-approved informed consent for the DSM265 and Ugandan studies included the consent to publish.
Ethics, consent and permissions
The DSM265 study was reviewed and approved by the Fred Hutchinson Cancer Research Center IRB (#8408). The study in Uganda was reviewed and approved by the University of California San Francisco IRB (#11-05995) and Uganda IRB (#2011-0167). In all studies, subjects were enrolled in the study only after obtaining informed consent using IRB-approved documents.
Funding was from the Bill and Melinda Gates Foundation for Plasmodium molecular diagnostic test development (OPP1133622 to S. Murphy). The DSM265 study clinical trial was supported by the Assistant Secretary of Defense for Health Affairs, through the Peer Reviewed Medical Research Program (PRMRP) under Award W81XWH-15-2-0010 and the Medicines for Malaria Venture. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the Department of Defense. The Ugandan field samples were funded in part by the National Institutes of Health as part of the International Centers of Excellence in Malaria Research program (U19AI089674).
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