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A powerful qPCR-high resolution melting assay with taqman probe in plasmodium species differentiation
BACKGROUND: The use of highly sensitive molecular tools in malaria diagnosis is currently largely restricted to research and epidemiological settings, but will ultimately be essential during elimination and potentially eradication. Accurate diagnosis and differentiation down to species levels, inclu...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916309/ https://www.ncbi.nlm.nih.gov/pubmed/33639949 http://dx.doi.org/10.1186/s12936-021-03662-w |
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author | Lamien-Meda, Aline Fuehrer, Hans-Peter Leitsch, David Noedl, Harald |
author_facet | Lamien-Meda, Aline Fuehrer, Hans-Peter Leitsch, David Noedl, Harald |
author_sort | Lamien-Meda, Aline |
collection | PubMed |
description | BACKGROUND: The use of highly sensitive molecular tools in malaria diagnosis is currently largely restricted to research and epidemiological settings, but will ultimately be essential during elimination and potentially eradication. Accurate diagnosis and differentiation down to species levels, including the two Plasmodium ovale species and zoonotic variants of the disease, will be important for the understanding of changing epidemiological patterns of the disease. METHODS: A qPCR-high resolution melting (HRM) method was to detect and differentiate all human Plasmodium species with one forward and one reverse primer set. The HRM detection method was further refined using a hydrolysis probe to specifically discriminate Plasmodium falciparum. RESULTS: Out of the 113 samples tested with the developed HRM-qPCR- P. falciparum probe assay, 96 (85.0 %) single infections, 12 (10.6 %) mixed infections, and 5 (4.4 %) were Plasmodium negative. The results were concordant with those of the nested PCR at 98.2 %. The assay limit of detection was varied from 21.47 to 46.43 copies /µl, equivalent to 1–2.11 parasites/µl. All P. falciparum infections were confirmed with the associated Taqman probe. CONCLUSIONS: Although the dependence on qPCR currently limits its deployment in resource-limited environments, this assay is highly sensitive and specific, easy to perform and convenient for Plasmodium mono-infection and may provide a novel tool for rapid and accurate malaria diagnosis also in epidemiological studies. |
format | Online Article Text |
id | pubmed-7916309 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-79163092021-03-02 A powerful qPCR-high resolution melting assay with taqman probe in plasmodium species differentiation Lamien-Meda, Aline Fuehrer, Hans-Peter Leitsch, David Noedl, Harald Malar J Research BACKGROUND: The use of highly sensitive molecular tools in malaria diagnosis is currently largely restricted to research and epidemiological settings, but will ultimately be essential during elimination and potentially eradication. Accurate diagnosis and differentiation down to species levels, including the two Plasmodium ovale species and zoonotic variants of the disease, will be important for the understanding of changing epidemiological patterns of the disease. METHODS: A qPCR-high resolution melting (HRM) method was to detect and differentiate all human Plasmodium species with one forward and one reverse primer set. The HRM detection method was further refined using a hydrolysis probe to specifically discriminate Plasmodium falciparum. RESULTS: Out of the 113 samples tested with the developed HRM-qPCR- P. falciparum probe assay, 96 (85.0 %) single infections, 12 (10.6 %) mixed infections, and 5 (4.4 %) were Plasmodium negative. The results were concordant with those of the nested PCR at 98.2 %. The assay limit of detection was varied from 21.47 to 46.43 copies /µl, equivalent to 1–2.11 parasites/µl. All P. falciparum infections were confirmed with the associated Taqman probe. CONCLUSIONS: Although the dependence on qPCR currently limits its deployment in resource-limited environments, this assay is highly sensitive and specific, easy to perform and convenient for Plasmodium mono-infection and may provide a novel tool for rapid and accurate malaria diagnosis also in epidemiological studies. BioMed Central 2021-02-28 /pmc/articles/PMC7916309/ /pubmed/33639949 http://dx.doi.org/10.1186/s12936-021-03662-w Text en © The Author(s) 2021 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Lamien-Meda, Aline Fuehrer, Hans-Peter Leitsch, David Noedl, Harald A powerful qPCR-high resolution melting assay with taqman probe in plasmodium species differentiation |
title | A powerful qPCR-high resolution melting assay with taqman probe in plasmodium species differentiation |
title_full | A powerful qPCR-high resolution melting assay with taqman probe in plasmodium species differentiation |
title_fullStr | A powerful qPCR-high resolution melting assay with taqman probe in plasmodium species differentiation |
title_full_unstemmed | A powerful qPCR-high resolution melting assay with taqman probe in plasmodium species differentiation |
title_short | A powerful qPCR-high resolution melting assay with taqman probe in plasmodium species differentiation |
title_sort | powerful qpcr-high resolution melting assay with taqman probe in plasmodium species differentiation |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916309/ https://www.ncbi.nlm.nih.gov/pubmed/33639949 http://dx.doi.org/10.1186/s12936-021-03662-w |
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