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

摘要

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.