Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites.

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...

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Autores principales:	Renate Schneider, Aline Lamien-Meda, Herbert Auer, Ursula Wiedermann-Schmidt, Peter L Chiodini, Julia Walochnik
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/458375b2380e44959fa1abaec23df227
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spelling	oai:doaj.org-article:458375b2380e44959fa1abaec23df2272021-12-02T20:11:01ZValidation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites.1932-620310.1371/journal.pone.0252887https://doaj.org/article/458375b2380e44959fa1abaec23df2272021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0252887https://doaj.org/toc/1932-6203Increasing 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 P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. 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 P. falciparum, is a significant advantage, especially if low-level parasitemia has to be ruled out. Even mixed infections of P. falciparum with P. vivax or P. ovale, respectively, were detected. In contrast to many other real-time PCR protocols, this novel FRET-qPCR allows the quantitative and species-specific detection of Plasmodium spp. in one single run. Solely, P. knowlesi was detected but could not be differentiated from P. vivax. 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.Renate SchneiderAline Lamien-MedaHerbert AuerUrsula Wiedermann-SchmidtPeter L ChiodiniJulia WalochnikPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 6, p e0252887 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Renate Schneider Aline Lamien-Meda Herbert Auer Ursula Wiedermann-Schmidt Peter L Chiodini Julia Walochnik Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites.
description	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 P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. 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 P. falciparum, is a significant advantage, especially if low-level parasitemia has to be ruled out. Even mixed infections of P. falciparum with P. vivax or P. ovale, respectively, were detected. In contrast to many other real-time PCR protocols, this novel FRET-qPCR allows the quantitative and species-specific detection of Plasmodium spp. in one single run. Solely, P. knowlesi was detected but could not be differentiated from P. vivax. 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.
format	article
author	Renate Schneider Aline Lamien-Meda Herbert Auer Ursula Wiedermann-Schmidt Peter L Chiodini Julia Walochnik
author_facet	Renate Schneider Aline Lamien-Meda Herbert Auer Ursula Wiedermann-Schmidt Peter L Chiodini Julia Walochnik
author_sort	Renate Schneider
title	Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites.
title_short	Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites.
title_full	Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites.
title_fullStr	Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites.
title_full_unstemmed	Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites.
title_sort	validation of a novel fret real-time pcr assay for simultaneous quantitative detection and discrimination of human plasmodium parasites.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/458375b2380e44959fa1abaec23df227
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Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites.

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