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Title: Recombinase polymerase amplification assay for rapid diagnostics of dengue infection
Authors: Ahmed Abd El Wahed
Pranav Patel
Oumar Faye
Sasikanya Thaloengsok
Doris Heidenreich
Ponpan Matangkasombut
Khajohnpong Manopwisedjaroen
Anavaj Sakuntabhai
Amadou A. Sall
Frank T. Hufert
Manfred Weidmann
Deutsches Primatenzentrum
Mansoura University
Robert Koch Institut
Institut Pasteur de Dakar
Mahidol University
Universitatsmedizin Gottingen
Institut Pasteur, Paris
Brandenburg Medical School Theodor Fontane
University of Stirling
Keywords: Agricultural and Biological Sciences;Biochemistry, Genetics and Molecular Biology
Issue Date: 15-Jun-2015
Citation: PLoS ONE. Vol.10, No.6 (2015)
Abstract: © 2015 Abd El Wahed et al. 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 credited. 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.
ISSN: 19326203
Appears in Collections:Scopus 2011-2015

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