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Characterization of Plasmodium ovale curtisi and P. ovale wallikeri in Western Kenya utilizing a novel species-specific real-time PCR assay.

Miller RH, Obuya CO, Wanja EW, Ogutu B, Waitumbi J, Luckhart S, Stewart VA - PLoS Negl Trop Dis (2015)

Bottom Line: Although P. ovale subspecies are similar based on morphology and geographical distribution, allelic differences indicate that P. ovale curtisi and P. ovale wallikeri are genetically divergent.Our P. ovale rbp2 qPCR assay validation experiments demonstrated a linear dynamic range from 6.25 rbp2 plasmid copies/microliter to 100,000 rbp2 plasmid copies/microliter and a limit of detection of 1.5 rbp2 plasmid copies/microliter.We identified P. ovale curtisi and P. ovale wallikeri in Western Kenya by DNA sequencing of the tryptophan-rich antigen gene, the small subunit ribosomal RNA gene, and the rbp2 gene.

View Article: PubMed Central - PubMed

Affiliation: Preventive Medicine and Biometrics, Uniformed Services University, Bethesda, Maryland, United States of America.

ABSTRACT

Background: Plasmodium ovale is comprised of two genetically distinct subspecies, P. ovale curtisi and P. ovale wallikeri. Although P. ovale subspecies are similar based on morphology and geographical distribution, allelic differences indicate that P. ovale curtisi and P. ovale wallikeri are genetically divergent. Additionally, potential clinical and latency duration differences between P. ovale curtisi and P. ovale wallikeri demonstrate the need for investigation into the contribution of this neglected malaria parasite to the global malaria burden.

Methods: In order to detect all P. ovale subspecies simultaneously, we developed an inclusive P. ovale-specific real-time PCR assay based on conserved regions between P. ovale curtisi and P. ovale wallikeri in the reticulocyte binding protein 2 (rbp2) gene. Additionally, we characterized the P. ovale subspecies prevalence from 22 asymptomatic malaria infections using multilocus genotyping to discriminate P. ovale curtisi and P. ovale wallikeri.

Results: Our P. ovale rbp2 qPCR assay validation experiments demonstrated a linear dynamic range from 6.25 rbp2 plasmid copies/microliter to 100,000 rbp2 plasmid copies/microliter and a limit of detection of 1.5 rbp2 plasmid copies/microliter. Specificity experiments showed the ability of the rbp2 qPCR assay to detect low-levels of P. ovale in the presence of additional malaria parasite species, including P. falciparum, P. vivax, and P. malariae. We identified P. ovale curtisi and P. ovale wallikeri in Western Kenya by DNA sequencing of the tryptophan-rich antigen gene, the small subunit ribosomal RNA gene, and the rbp2 gene.

Conclusions: Our novel P. ovale rbp2 qPCR assay detects P. ovale curtisi and P. ovale wallikeri simultaneously and can be utilized to characterize the prevalence, distribution, and burden of P. ovale in malaria endemic regions. Using multilocus genotyping, we also provided the first description of the prevalence of P. ovale curtisi and P. ovale wallikeri in Western Kenya, a region holoendemic for malaria transmission.

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P. ovale reticulocyte binding protein 2 (rbp2) sequence alignment.The P. ovale curtisi (GU813971) and P. ovale wallikeri (GU813972) rbp2 sequences were aligned using EMBL-EBI Clustal Omega program and visualized in Jalview with the default Jalview nucleotide color scheme (green for adenine, orange for cytosine, red for guanine, and blue for thymine). Primers and probe were designed based on conserved regions between the two P. ovale subspecies. The forward (PoRBP2fwd1) and reverse (PoRBP2rev1) primers are indicated by arrows and the hydrolysis probe (PoRBP2p) binding site (boxed) is located in between the forward and reverse primer.
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pntd.0003469.g001: P. ovale reticulocyte binding protein 2 (rbp2) sequence alignment.The P. ovale curtisi (GU813971) and P. ovale wallikeri (GU813972) rbp2 sequences were aligned using EMBL-EBI Clustal Omega program and visualized in Jalview with the default Jalview nucleotide color scheme (green for adenine, orange for cytosine, red for guanine, and blue for thymine). Primers and probe were designed based on conserved regions between the two P. ovale subspecies. The forward (PoRBP2fwd1) and reverse (PoRBP2rev1) primers are indicated by arrows and the hydrolysis probe (PoRBP2p) binding site (boxed) is located in between the forward and reverse primer.

Mentions: Reticulocyte binding protein 2 (rbp2) gene. The reticulocyte binding protein 2 (rbp2) gene was utilized by Oguike et al. 2011 to differentiate between P. ovale subspecies using qPCR melt curve profiles based on six SNPs present within a 120 base pair fragment. We designed a novel set of primers (Table 1, IDT) using Primer Express software (Life Technologies, version 3.0; Frederick, MD, USA) to amplify a smaller, 74 base pair region of the rbp2 gene for assay development. Our primers (PoRBP2f and PoRBP2r) are located within conserved DNA sequences of the P. ovale subspecies to ensure detection and amplification of both P. ovale subspecies. The amplicon also contains a single SNP to distinguish P. ovale subspecies by DNA sequencing. Fig. 1 shows the single SNP in the rbp2 amplicon at position 431, in which P. ovale curtisi contains an adenine and P. ovale wallikeri contains a thymine. Primer BLAST was utilized to ensure our primers were specific for P. ovale and would not amplify non- P. ovale malaria parasite DNA or human DNA. PCRs consisted of 1X Sigma JumpStart REDTaq ReadyMix Reaction Mix, 25 picomoles of each primer, and one microliter of template, with a final volume of 25 microliters. PCR cycling conditions were as follows: initial denaturation at 95°C for 2 minutes followed by 40 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 10 minutes.


Characterization of Plasmodium ovale curtisi and P. ovale wallikeri in Western Kenya utilizing a novel species-specific real-time PCR assay.

Miller RH, Obuya CO, Wanja EW, Ogutu B, Waitumbi J, Luckhart S, Stewart VA - PLoS Negl Trop Dis (2015)

P. ovale reticulocyte binding protein 2 (rbp2) sequence alignment.The P. ovale curtisi (GU813971) and P. ovale wallikeri (GU813972) rbp2 sequences were aligned using EMBL-EBI Clustal Omega program and visualized in Jalview with the default Jalview nucleotide color scheme (green for adenine, orange for cytosine, red for guanine, and blue for thymine). Primers and probe were designed based on conserved regions between the two P. ovale subspecies. The forward (PoRBP2fwd1) and reverse (PoRBP2rev1) primers are indicated by arrows and the hydrolysis probe (PoRBP2p) binding site (boxed) is located in between the forward and reverse primer.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4295880&req=5

pntd.0003469.g001: P. ovale reticulocyte binding protein 2 (rbp2) sequence alignment.The P. ovale curtisi (GU813971) and P. ovale wallikeri (GU813972) rbp2 sequences were aligned using EMBL-EBI Clustal Omega program and visualized in Jalview with the default Jalview nucleotide color scheme (green for adenine, orange for cytosine, red for guanine, and blue for thymine). Primers and probe were designed based on conserved regions between the two P. ovale subspecies. The forward (PoRBP2fwd1) and reverse (PoRBP2rev1) primers are indicated by arrows and the hydrolysis probe (PoRBP2p) binding site (boxed) is located in between the forward and reverse primer.
Mentions: Reticulocyte binding protein 2 (rbp2) gene. The reticulocyte binding protein 2 (rbp2) gene was utilized by Oguike et al. 2011 to differentiate between P. ovale subspecies using qPCR melt curve profiles based on six SNPs present within a 120 base pair fragment. We designed a novel set of primers (Table 1, IDT) using Primer Express software (Life Technologies, version 3.0; Frederick, MD, USA) to amplify a smaller, 74 base pair region of the rbp2 gene for assay development. Our primers (PoRBP2f and PoRBP2r) are located within conserved DNA sequences of the P. ovale subspecies to ensure detection and amplification of both P. ovale subspecies. The amplicon also contains a single SNP to distinguish P. ovale subspecies by DNA sequencing. Fig. 1 shows the single SNP in the rbp2 amplicon at position 431, in which P. ovale curtisi contains an adenine and P. ovale wallikeri contains a thymine. Primer BLAST was utilized to ensure our primers were specific for P. ovale and would not amplify non- P. ovale malaria parasite DNA or human DNA. PCRs consisted of 1X Sigma JumpStart REDTaq ReadyMix Reaction Mix, 25 picomoles of each primer, and one microliter of template, with a final volume of 25 microliters. PCR cycling conditions were as follows: initial denaturation at 95°C for 2 minutes followed by 40 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 10 minutes.

Bottom Line: Although P. ovale subspecies are similar based on morphology and geographical distribution, allelic differences indicate that P. ovale curtisi and P. ovale wallikeri are genetically divergent.Our P. ovale rbp2 qPCR assay validation experiments demonstrated a linear dynamic range from 6.25 rbp2 plasmid copies/microliter to 100,000 rbp2 plasmid copies/microliter and a limit of detection of 1.5 rbp2 plasmid copies/microliter.We identified P. ovale curtisi and P. ovale wallikeri in Western Kenya by DNA sequencing of the tryptophan-rich antigen gene, the small subunit ribosomal RNA gene, and the rbp2 gene.

View Article: PubMed Central - PubMed

Affiliation: Preventive Medicine and Biometrics, Uniformed Services University, Bethesda, Maryland, United States of America.

ABSTRACT

Background: Plasmodium ovale is comprised of two genetically distinct subspecies, P. ovale curtisi and P. ovale wallikeri. Although P. ovale subspecies are similar based on morphology and geographical distribution, allelic differences indicate that P. ovale curtisi and P. ovale wallikeri are genetically divergent. Additionally, potential clinical and latency duration differences between P. ovale curtisi and P. ovale wallikeri demonstrate the need for investigation into the contribution of this neglected malaria parasite to the global malaria burden.

Methods: In order to detect all P. ovale subspecies simultaneously, we developed an inclusive P. ovale-specific real-time PCR assay based on conserved regions between P. ovale curtisi and P. ovale wallikeri in the reticulocyte binding protein 2 (rbp2) gene. Additionally, we characterized the P. ovale subspecies prevalence from 22 asymptomatic malaria infections using multilocus genotyping to discriminate P. ovale curtisi and P. ovale wallikeri.

Results: Our P. ovale rbp2 qPCR assay validation experiments demonstrated a linear dynamic range from 6.25 rbp2 plasmid copies/microliter to 100,000 rbp2 plasmid copies/microliter and a limit of detection of 1.5 rbp2 plasmid copies/microliter. Specificity experiments showed the ability of the rbp2 qPCR assay to detect low-levels of P. ovale in the presence of additional malaria parasite species, including P. falciparum, P. vivax, and P. malariae. We identified P. ovale curtisi and P. ovale wallikeri in Western Kenya by DNA sequencing of the tryptophan-rich antigen gene, the small subunit ribosomal RNA gene, and the rbp2 gene.

Conclusions: Our novel P. ovale rbp2 qPCR assay detects P. ovale curtisi and P. ovale wallikeri simultaneously and can be utilized to characterize the prevalence, distribution, and burden of P. ovale in malaria endemic regions. Using multilocus genotyping, we also provided the first description of the prevalence of P. ovale curtisi and P. ovale wallikeri in Western Kenya, a region holoendemic for malaria transmission.

Show MeSH
Related in: MedlinePlus