A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi
Background: The misdiagnosis of Plasmodium knowlesi by microscopy has prompted a re-evaluation of the geographic distribution, prevalence and pathogenesis of this species using molecular diagnostic tools. In this report, a specific probe for P. knowlesi, that can be used in a previously described...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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BioMed Central Ltd.
2010
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| Online Access: | http://ir.unimas.my/id/eprint/15804/ http://ir.unimas.my/id/eprint/15804/1/A%20TaqMan%20real-time%20PCR%20assay%20for%20the%20detection%20%28abstract%29.pdf |
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| author | Paul Cliff, Simon Divis Shokoples, Sandra E Balbir, Singh Yanow, Stephanie K.Y |
| author_facet | Paul Cliff, Simon Divis Shokoples, Sandra E Balbir, Singh Yanow, Stephanie K.Y |
| author_sort | Paul Cliff, Simon Divis |
| building | UNIMAS Institutional Repository |
| collection | Online Access |
| description | Background: The misdiagnosis of Plasmodium knowlesi by microscopy has prompted a re-evaluation of the
geographic distribution, prevalence and pathogenesis of this species using molecular diagnostic tools. In this
report, a specific probe for P. knowlesi, that can be used in a previously described TaqMan real-time PCR assay for
detection of Plasmodium spp., and Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and
Plasmodium ovale, was designed and validated against clinical samples.
Methods: A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within
the P. knowlesi small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were
determined using plasmids containing P. knowlesi DNA and genomic DNA of P. falciparum, P. knowlesi, P. malariae,
P. ovale and P. vivax isolated from clinical samples. DNA samples of the simian malaria parasites Plasmodium
cynomolgi and Plasmodium inui that can infect humans under experimental conditions were also examined
together with human DNA samples.
Results: Analytical sensitivity of the P. knowlesi-specific assay was 10 copies/μL and quantitation was linear over a
range of 10-106 copies. The sensitivity of the assay is equivalent to nested PCR and P. knowlesi DNA was detected
from all 40 clinical P. knowlesi specimens, including one from a patient with a parasitaemia of three parasites/μL of
blood. No cross-reactivity was observed with 67 Plasmodium DNA samples (31 P. falciparum, 23 P. vivax, six P. ovale,
three P. malariae, one P. malariae/P. ovale, one P. falciparum/P. malariae, one P. inui and one P. cynomolgi) and four
samples of human DNA.
Conclusions: This test demonstrated excellent sensitivity and specificity, and adds P. knowlesi to the repertoire of
Plasmodium targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA
copy number provides a useful advantage over other molecular assays to investigate the correlation between
levels of infection and the spectrum of disease. |
| first_indexed | 2025-11-15T06:47:28Z |
| format | Article |
| id | unimas-15804 |
| institution | Universiti Malaysia Sarawak |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-15T06:47:28Z |
| publishDate | 2010 |
| publisher | BioMed Central Ltd. |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | unimas-158042017-04-04T03:47:25Z http://ir.unimas.my/id/eprint/15804/ A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi Paul Cliff, Simon Divis Shokoples, Sandra E Balbir, Singh Yanow, Stephanie K.Y RZ Other systems of medicine Background: The misdiagnosis of Plasmodium knowlesi by microscopy has prompted a re-evaluation of the geographic distribution, prevalence and pathogenesis of this species using molecular diagnostic tools. In this report, a specific probe for P. knowlesi, that can be used in a previously described TaqMan real-time PCR assay for detection of Plasmodium spp., and Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, was designed and validated against clinical samples. Methods: A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the P. knowlesi small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing P. knowlesi DNA and genomic DNA of P. falciparum, P. knowlesi, P. malariae, P. ovale and P. vivax isolated from clinical samples. DNA samples of the simian malaria parasites Plasmodium cynomolgi and Plasmodium inui that can infect humans under experimental conditions were also examined together with human DNA samples. Results: Analytical sensitivity of the P. knowlesi-specific assay was 10 copies/μL and quantitation was linear over a range of 10-106 copies. The sensitivity of the assay is equivalent to nested PCR and P. knowlesi DNA was detected from all 40 clinical P. knowlesi specimens, including one from a patient with a parasitaemia of three parasites/μL of blood. No cross-reactivity was observed with 67 Plasmodium DNA samples (31 P. falciparum, 23 P. vivax, six P. ovale, three P. malariae, one P. malariae/P. ovale, one P. falciparum/P. malariae, one P. inui and one P. cynomolgi) and four samples of human DNA. Conclusions: This test demonstrated excellent sensitivity and specificity, and adds P. knowlesi to the repertoire of Plasmodium targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA copy number provides a useful advantage over other molecular assays to investigate the correlation between levels of infection and the spectrum of disease. BioMed Central Ltd. 2010 Article PeerReviewed text en http://ir.unimas.my/id/eprint/15804/1/A%20TaqMan%20real-time%20PCR%20assay%20for%20the%20detection%20%28abstract%29.pdf Paul Cliff, Simon Divis and Shokoples, Sandra E and Balbir, Singh and Yanow, Stephanie K.Y (2010) A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi. Malaria Journal, 9 (344). ISSN 14752875 https://malariajournal.biomedcentral.com/articles/10.1186/1475-2875-9-344 DOI: 10.1186/1475-2875-9-344 |
| spellingShingle | RZ Other systems of medicine Paul Cliff, Simon Divis Shokoples, Sandra E Balbir, Singh Yanow, Stephanie K.Y A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi |
| title | A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi |
| title_full | A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi |
| title_fullStr | A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi |
| title_full_unstemmed | A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi |
| title_short | A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi |
| title_sort | taqman real-time pcr assay for the detection and quantitation of plasmodium knowlesi |
| topic | RZ Other systems of medicine |
| url | http://ir.unimas.my/id/eprint/15804/ http://ir.unimas.my/id/eprint/15804/ http://ir.unimas.my/id/eprint/15804/ http://ir.unimas.my/id/eprint/15804/1/A%20TaqMan%20real-time%20PCR%20assay%20for%20the%20detection%20%28abstract%29.pdf |