Cargando…
Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants
BACKGROUND: A simple real-time PCR assay using one set of primer and probe for rapid, sensitive and quantitative detection of Plasmodium species, with simultaneous differentiation of Plasmodium falciparum from the three other Plasmodium species (Plasmodium vivax, Plasmodium ovale and Plasmodium mala...
Autores principales: | , , , , , , , , , , |
---|---|
Formato: | Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2008
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2386128/ https://www.ncbi.nlm.nih.gov/pubmed/18442362 http://dx.doi.org/10.1186/1475-2875-7-70 |
_version_ | 1782155216517332992 |
---|---|
author | Safeukui, Innocent Millet, Pascal Boucher, Sébastien Melinard, Laurence Fregeville, Frédéric Receveur, Marie-Catherine Pistone, Thierry Fialon, Pierre Vincendeau, Philippe Fleury, Hervé Malvy, Denis |
author_facet | Safeukui, Innocent Millet, Pascal Boucher, Sébastien Melinard, Laurence Fregeville, Frédéric Receveur, Marie-Catherine Pistone, Thierry Fialon, Pierre Vincendeau, Philippe Fleury, Hervé Malvy, Denis |
author_sort | Safeukui, Innocent |
collection | PubMed |
description | BACKGROUND: A simple real-time PCR assay using one set of primer and probe for rapid, sensitive and quantitative detection of Plasmodium species, with simultaneous differentiation of Plasmodium falciparum from the three other Plasmodium species (Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) in febrile returning travellers and migrants was developed and evaluated. METHODS: Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be perfect matches to the 18S rRNA gene of the fourth Plasmodium species, while the acceptor probe sequence was designed for P. falciparum over a region containing one mismatched, which allowed differentiation of the three other Plasmodium species. The performance characteristics of the real-time PCR assay were compared with those of conventional PCR and microscopy-based diagnosis from 119 individuals with a suspected clinical diagnostic of imported malaria. RESULTS: Blood samples with parasite densities less than 0.01% were all detected, and analytical sensitivity was 0.5 parasite per PCR reaction. The melt curve means Tms (standard deviation) in clinical isolates were 60.5°C (0.6°C) for P. falciparum infection and 64.6°C (1.8°C) for non-P. falciparum species. These Tms values of the P. falciparum or non-P. falciparum species did not vary with the geographic origin of the parasite. The real-time PCR results correlated with conventional PCR using both genus-specific (Kappa coefficient: 0.95, 95% confidence interval: 0.9 – 1) or P. falciparum-specific (0.91, 0.8 – 1) primers, or with the microscopy results (0.70, 0.6 – 0.8). The real-time assay was 100% sensitive and specific for differentiation of P. falciparum to non-P. falciparum species, compared with conventional PCR or microscopy. The real-time PCR assay can also detect individuals with mixed infections (P. falciparum and non-P. falciparum sp.) in the same sample. CONCLUSION: This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of P. falciparum to other Plasmodium species. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed. |
format | Text |
id | pubmed-2386128 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-23861282008-05-15 Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants Safeukui, Innocent Millet, Pascal Boucher, Sébastien Melinard, Laurence Fregeville, Frédéric Receveur, Marie-Catherine Pistone, Thierry Fialon, Pierre Vincendeau, Philippe Fleury, Hervé Malvy, Denis Malar J Methodology BACKGROUND: A simple real-time PCR assay using one set of primer and probe for rapid, sensitive and quantitative detection of Plasmodium species, with simultaneous differentiation of Plasmodium falciparum from the three other Plasmodium species (Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) in febrile returning travellers and migrants was developed and evaluated. METHODS: Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be perfect matches to the 18S rRNA gene of the fourth Plasmodium species, while the acceptor probe sequence was designed for P. falciparum over a region containing one mismatched, which allowed differentiation of the three other Plasmodium species. The performance characteristics of the real-time PCR assay were compared with those of conventional PCR and microscopy-based diagnosis from 119 individuals with a suspected clinical diagnostic of imported malaria. RESULTS: Blood samples with parasite densities less than 0.01% were all detected, and analytical sensitivity was 0.5 parasite per PCR reaction. The melt curve means Tms (standard deviation) in clinical isolates were 60.5°C (0.6°C) for P. falciparum infection and 64.6°C (1.8°C) for non-P. falciparum species. These Tms values of the P. falciparum or non-P. falciparum species did not vary with the geographic origin of the parasite. The real-time PCR results correlated with conventional PCR using both genus-specific (Kappa coefficient: 0.95, 95% confidence interval: 0.9 – 1) or P. falciparum-specific (0.91, 0.8 – 1) primers, or with the microscopy results (0.70, 0.6 – 0.8). The real-time assay was 100% sensitive and specific for differentiation of P. falciparum to non-P. falciparum species, compared with conventional PCR or microscopy. The real-time PCR assay can also detect individuals with mixed infections (P. falciparum and non-P. falciparum sp.) in the same sample. CONCLUSION: This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of P. falciparum to other Plasmodium species. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed. BioMed Central 2008-04-28 /pmc/articles/PMC2386128/ /pubmed/18442362 http://dx.doi.org/10.1186/1475-2875-7-70 Text en Copyright © 2008 Safeukui et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methodology Safeukui, Innocent Millet, Pascal Boucher, Sébastien Melinard, Laurence Fregeville, Frédéric Receveur, Marie-Catherine Pistone, Thierry Fialon, Pierre Vincendeau, Philippe Fleury, Hervé Malvy, Denis Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants |
title | Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants |
title_full | Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants |
title_fullStr | Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants |
title_full_unstemmed | Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants |
title_short | Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants |
title_sort | evaluation of fret real-time pcr assay for rapid detection and differentiation of plasmodium species in returning travellers and migrants |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2386128/ https://www.ncbi.nlm.nih.gov/pubmed/18442362 http://dx.doi.org/10.1186/1475-2875-7-70 |
work_keys_str_mv | AT safeukuiinnocent evaluationoffretrealtimepcrassayforrapiddetectionanddifferentiationofplasmodiumspeciesinreturningtravellersandmigrants AT milletpascal evaluationoffretrealtimepcrassayforrapiddetectionanddifferentiationofplasmodiumspeciesinreturningtravellersandmigrants AT bouchersebastien evaluationoffretrealtimepcrassayforrapiddetectionanddifferentiationofplasmodiumspeciesinreturningtravellersandmigrants AT melinardlaurence evaluationoffretrealtimepcrassayforrapiddetectionanddifferentiationofplasmodiumspeciesinreturningtravellersandmigrants AT fregevillefrederic evaluationoffretrealtimepcrassayforrapiddetectionanddifferentiationofplasmodiumspeciesinreturningtravellersandmigrants AT receveurmariecatherine evaluationoffretrealtimepcrassayforrapiddetectionanddifferentiationofplasmodiumspeciesinreturningtravellersandmigrants AT pistonethierry evaluationoffretrealtimepcrassayforrapiddetectionanddifferentiationofplasmodiumspeciesinreturningtravellersandmigrants AT fialonpierre evaluationoffretrealtimepcrassayforrapiddetectionanddifferentiationofplasmodiumspeciesinreturningtravellersandmigrants AT vincendeauphilippe evaluationoffretrealtimepcrassayforrapiddetectionanddifferentiationofplasmodiumspeciesinreturningtravellersandmigrants AT fleuryherve evaluationoffretrealtimepcrassayforrapiddetectionanddifferentiationofplasmodiumspeciesinreturningtravellersandmigrants AT malvydenis evaluationoffretrealtimepcrassayforrapiddetectionanddifferentiationofplasmodiumspeciesinreturningtravellersandmigrants |