Early detection of dengue virus by use of reverse transcription-recombinase polymerase amplification

• Main Authors: Teoh, Boon-Teong, Sam, Sing-Sin, Tan, Kim-Kee, Danlami, Mohammed Bashar, Shu, Meng-Hooi, Johari, Jefree, Hooi, Poh-Sim, Brooks, David, Piepenburg, Olaf, Nentwich, Oliver, Wilder-Smith, Annelies, Franco, Leticia, Tenorio, Antonio, AbuBakar, Sazaly
• Other Authors: Tang, Y.-W.
• Format: Article
• Language: English
• Published: 2015
• Subjects: DRNTU::Science::Biological sciences::Microbiology
• Online Access: https://hdl.handle.net/10356/101099; http://hdl.handle.net/10220/25718
• Institution: Nanyang Technological University

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 min 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 serum samples of patients with clinically suspected dengue. The sera were simultaneously tested for DENV using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay, quantitative RT-PCR (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 of ≥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.