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Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection
BACKGROUND: Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR) are the standard method for molecular detection of the dengue vi...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468249/ https://www.ncbi.nlm.nih.gov/pubmed/26075598 http://dx.doi.org/10.1371/journal.pone.0129682 |
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author | Abd El Wahed, Ahmed Patel, Pranav Faye, Oumar Thaloengsok, Sasikanya Heidenreich, Doris Matangkasombut, Ponpan Manopwisedjaroen, Khajohnpong Sakuntabhai, Anavaj Sall, Amadou A. Hufert, Frank T. Weidmann, Manfred |
author_facet | Abd El Wahed, Ahmed Patel, Pranav Faye, Oumar Thaloengsok, Sasikanya Heidenreich, Doris Matangkasombut, Ponpan Manopwisedjaroen, Khajohnpong Sakuntabhai, Anavaj Sall, Amadou A. Hufert, Frank T. Weidmann, Manfred |
author_sort | Abd El Wahed, Ahmed |
collection | PubMed |
description | BACKGROUND: Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR) are the standard method for molecular detection of the dengue virus (DENV). Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA) assays were developed to detect DENV1-4. METHODOLOGY/PRINCIPAL FINDINGS: Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4) to 241 (DENV1-3) RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal) and in Bangkok (Thailand). In Kedougou, the RT-RPA was operated at an ambient temperature of 38°C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31) and 100% (n=23), respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90) and 100%(n=41), respectively. CONCLUSIONS/SIGNIFICANCE: During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations. |
format | Online Article Text |
id | pubmed-4468249 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-44682492015-06-25 Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection Abd El Wahed, Ahmed Patel, Pranav Faye, Oumar Thaloengsok, Sasikanya Heidenreich, Doris Matangkasombut, Ponpan Manopwisedjaroen, Khajohnpong Sakuntabhai, Anavaj Sall, Amadou A. Hufert, Frank T. Weidmann, Manfred PLoS One Research Article BACKGROUND: Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR) are the standard method for molecular detection of the dengue virus (DENV). Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA) assays were developed to detect DENV1-4. METHODOLOGY/PRINCIPAL FINDINGS: Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4) to 241 (DENV1-3) RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal) and in Bangkok (Thailand). In Kedougou, the RT-RPA was operated at an ambient temperature of 38°C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31) and 100% (n=23), respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90) and 100%(n=41), respectively. CONCLUSIONS/SIGNIFICANCE: During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations. Public Library of Science 2015-06-15 /pmc/articles/PMC4468249/ /pubmed/26075598 http://dx.doi.org/10.1371/journal.pone.0129682 Text en © 2015 Abd El Wahed et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Abd El Wahed, Ahmed Patel, Pranav Faye, Oumar Thaloengsok, Sasikanya Heidenreich, Doris Matangkasombut, Ponpan Manopwisedjaroen, Khajohnpong Sakuntabhai, Anavaj Sall, Amadou A. Hufert, Frank T. Weidmann, Manfred Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection |
title | Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection |
title_full | Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection |
title_fullStr | Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection |
title_full_unstemmed | Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection |
title_short | Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection |
title_sort | recombinase polymerase amplification assay for rapid diagnostics of dengue infection |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468249/ https://www.ncbi.nlm.nih.gov/pubmed/26075598 http://dx.doi.org/10.1371/journal.pone.0129682 |
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