Detection of dengue virus in ten minutes using reverse transcription recombinase polymerase amplification assay

Abstract

Background: Over 2.5 billion are at risk fromdengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. The real-time reverse transcription polymerase chain reaction (RTPCR) is the standard formolecular detectionof denguevirus (DENV) in acute phase of DF. Real-time RT-PCR analysis is not suitable for on-site of outbreak screening. The PCR devices are large, expensive, and complex. In this study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed to detect DENV 1-4. Methods & Materials: RNA molecular standards comprised of the 3′nontranslated region of DENV 1-4 were prepared. The assay sensitivity was determined by analysis of eight assay runs of serial dilutions of the RNA molecular standard. The assay specificity was evaluated against a panel of viruses considered for differential diagnosis with DENV 1-4. The assay performance was validated by testing 31blood samples in comparison to real-timeRT-PCR. To test the possibility of using RT-RPA assays for outbreak investigations, a mobile RT-RPA unit was operated in Kedougou, Senegal. Results: The DENV 1-4 RT-RPA was rapid (3-10minutes). The analytical sensitivity was 100 and 10 RNA molecules for DENV 1-3 and DENV 4, respectively. No cross reactivity with other viruses causing similar clinical picture was observed. Both RT-RPA and RT-PCR assays showed comparable results (96% sensitivity). The RT-RPA assay was significantly quicker than the real-time RT-PCR assay (10minutes for RT-RPAand90minutes for real-timeRT-PCR). In a field study in Kedougou, Senegal, a magnetic-bead based total nucleic acid extraction method was combined with RT-RPA on a portable instrument (tubescanner). The RT-RPA assaywas successfully used to detect DENV RNA extracted from gamma-irradiation inactivated lyophilized virus supernatants. All operations were carried out at an ambient temperature of 38 ◦C with auxiliary electricity tapped from a vehicle. Conclusion: The developed DENV 1-4 RT-RPA assay constitutes a suitable accurate, sensitive, and rapid assay for DF diagnosis during the first 5 days of infection. Moreover, the use of a portable fluorescence-reading device extends its application potential to point-of-care use or at the outbreak site diagnosis.