Early detection of the dengue virus using reverse transcription-recombinase polymerase amplification

A method for the rapid diagnosis of early dengue virus (DENV) infection is highly needed. Here, a prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed. The assay detected DENV RNA in <20 minutes without the need for thermocycling amplification. The assay enabled the detection of as few as 10 copies of DENV RNA. The designed RT-RPA primers and exo-probe detected the DENV genome of at least 12 genotypes of DENV circulating globally without cross-reacting with other arboviruses. We assessed the diagnostic performance of the RT-RPA assay for the detection of DENV RNA in 203 sera of clinically suspected dengue patients. The sera were simultaneously tested for DENV using RT-loop-mediated isothermal amplification (RT-LAMP), quantitative RT-polymerase chain reaction (qRT-PCR), and IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). Acute DENV infection was confirmed in 130 samples and 61 of the samples (46.9%) were classified as viremic with qRT-PCR. The RT-RPA assay showed good concordance (κ ≥ 0.723) with the RT-LAMP and qRT-PCR assays in detecting the dengue viremic samples. When used in combination with ELISA, both the RT-RPA and RT-LAMP assays increased the detection of acute DENV infection to ≥95.7% (≥45/47) in samples obtained within 5 days of illness. The results from the study suggest that the RT-RPA assay is the most rapid molecular diagnostic tool available for the detection of DENV. Hence, it is possible to use the RT-RPA assay in a laboratory to complement routine serology testing for dengue. IMPORTANCE: Nucleic acid detection methods are gradually being accepted as diagnostic platforms for the detection of DENV, especially in well-equipped diagnostic facilities. Their application in low-resource settings, however, is limited mostly due to the high cost and skill required. In this study, a novel RT-RPA assay was developed for the detection of DENV. The sensitivity and specificity of the RT-RPA assay were comparable to those of the RT-LAMP and qRT-PCR methods. The RT-RPA assay is rapid and simple to perform without the need for costly equipment or a high level of skill. Hence, it has the potential to be implemented as a routine diagnostic tool at health care centers and clinics in endemic regions where early detection of viremic dengue patients is needed for better treatment and outbreak control measures.Accepted manuscript posted online 7 January 2015, Free via Open Access: OA. The authors have paid a fee to allow immediate free access to this article.