{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T04:21:49Z","timestamp":1777522909648,"version":"3.51.4"},"reference-count":23,"publisher":"Public Library of Science (PLoS)","issue":"6","license":[{"start":{"date-parts":[[2021,6,4]],"date-time":"2021-06-04T00:00:00Z","timestamp":1622764800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Increasing numbers of travelers returning from endemic areas, migrants, and refugees have led to a significant rise in the number of imported malaria cases in non-endemic countries. Real- time PCR serves as an excellent diagnostic tool, especially in regions where experience in microscopy is limited. A novel fluorescence resonance energy transfer-based real-time PCR (FRET-qPCR) was developed and evaluated using 56 reference samples of the United Kingdom National External Quality Assessment Service (UK NEQAS) for molecular detection of malaria, including <jats:italic>P<\/jats:italic>. <jats:italic>falciparum<\/jats:italic>, <jats:italic>P<\/jats:italic>. <jats:italic>vivax<\/jats:italic>, <jats:italic>P<\/jats:italic>. <jats:italic>ovale<\/jats:italic>, <jats:italic>P<\/jats:italic>. <jats:italic>malariae<\/jats:italic>, and <jats:italic>P<\/jats:italic>. <jats:italic>knowlesi<\/jats:italic>. Species identification is based on single nucleotide polymorphisms (SNPs) within the genome where the MalLC640 probe binds, lowering the melting temperature in the melting curve analysis. The novel FRET-qPCR achieved 100% (n = 56) correct results, compared to 96.43% performing nested PCR. The high sensitivity, with a calculated limit of detection of 199.97 parasites\/mL blood for <jats:italic>P<\/jats:italic>. <jats:italic>falciparum<\/jats:italic>, is a significant advantage, especially if low-level parasitemia has to be ruled out. Even mixed infections of <jats:italic>P<\/jats:italic>. <jats:italic>falciparum<\/jats:italic> with <jats:italic>P<\/jats:italic>. <jats:italic>vivax<\/jats:italic> or <jats:italic>P<\/jats:italic>. <jats:italic>ovale<\/jats:italic>, respectively, were detected. In contrast to many other real-time PCR protocols, this novel FRET-qPCR allows the quantitative and species-specific detection of <jats:italic>Plasmodium<\/jats:italic> spp. in one single run. Solely, <jats:italic>P<\/jats:italic>. <jats:italic>knowlesi<\/jats:italic> was detected but could not be differentiated from <jats:italic>P<\/jats:italic>. <jats:italic>vivax<\/jats:italic>. The turnaround time of this novel FRET-qPCR including DNA extraction is less than two hours, qualifying it for routine clinical applications, including treatment monitoring.<\/jats:p>","DOI":"10.1371\/journal.pone.0252887","type":"journal-article","created":{"date-parts":[[2021,6,4]],"date-time":"2021-06-04T18:51:42Z","timestamp":1622832702000},"page":"e0252887","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":13,"title":["Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium 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