{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T16:23:12Z","timestamp":1792686192656,"version":"4.5.0"},"reference-count":37,"publisher":"Springer Science and Business Media LLC","issue":"1","content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Malar J"],"published-print":{"date-parts":[[2010,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n          <jats:sec>\n            <jats:title>Background<\/jats:title>\n            <jats:p>The misdiagnosis of <jats:italic>Plasmodium knowlesi<\/jats:italic> 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 <jats:italic>P. knowlesi<\/jats:italic>, that can be used in a previously described TaqMan real-time PCR assay for detection of <jats:italic>Plasmodium<\/jats:italic> spp., and <jats:italic>Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae<\/jats:italic> and <jats:italic>Plasmodium ovale<\/jats:italic>, was designed and validated against clinical samples.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Methods<\/jats:title>\n            <jats:p>A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the <jats:italic>P. knowlesi<\/jats:italic> small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing <jats:italic>P. knowlesi<\/jats:italic> DNA and genomic DNA of <jats:italic>P. falciparum, P. knowlesi, P. malariae, P. ovale<\/jats:italic> and <jats:italic>P. vivax<\/jats:italic> isolated from clinical samples. DNA samples of the simian malaria parasites <jats:italic>Plasmodium cynomolgi<\/jats:italic> and <jats:italic>Plasmodium inui<\/jats:italic> that can infect humans under experimental conditions were also examined together with human DNA samples.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>Analytical sensitivity of the <jats:italic>P. knowlesi<\/jats:italic>-specific assay was 10 copies\/\u03bcL and quantitation was linear over a range of 10-10<jats:sup>6<\/jats:sup> copies. The sensitivity of the assay is equivalent to nested PCR and <jats:italic>P. knowlesi<\/jats:italic> DNA was detected from all 40 clinical <jats:italic>P. knowlesi<\/jats:italic> specimens, including one from a patient with a parasitaemia of three parasites\/\u03bcL of blood. No cross-reactivity was observed with 67 <jats:italic>Plasmodium<\/jats:italic> DNA samples (31 <jats:italic>P. falciparum<\/jats:italic>, 23 <jats:italic>P. vivax<\/jats:italic>, six <jats:italic>P. ovale<\/jats:italic>, three <jats:italic>P. malariae<\/jats:italic>, one <jats:italic>P. malariae\/P. ovale<\/jats:italic>, one <jats:italic>P. falciparum\/P. malariae, one P. inui and one P. cynomolgi)<\/jats:italic> and four samples of human DNA.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusions<\/jats:title>\n            <jats:p>This test demonstrated excellent sensitivity and specificity, and adds <jats:italic>P. knowlesi<\/jats:italic> to the repertoire of <jats:italic>Plasmodium<\/jats:italic> 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.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/1475-2875-9-344","type":"journal-article","created":{"date-parts":[[2010,11,30]],"date-time":"2010-11-30T19:15:05Z","timestamp":1291144505000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":76,"title":["A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi"],"prefix":"10.1186","volume":"9","author":[{"given":"Paul CS","family":"Divis","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandra E","family":"Shokoples","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Balbir","family":"Singh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephanie K","family":"Yanow","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2010,11,30]]},"reference":[{"key":"1426_CR1","doi-asserted-by":"publisher","first-page":"66","DOI":"10.1128\/CMR.15.1.66-78.2002","volume":"15","author":"A Moody","year":"2002","unstructured":"Moody A: Rapid diagnostic tests for malaria parasites. 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