Simultaneous detection of dengue virus serotypes in a dual-serotype-detection nucleic acid based lateral flow assay

Dengue virus (DENV) is an important arthropod-borne viral disease, with four antigenically and genetically diverse serotypes (DENV-1, DENV-2, DENV-3, and DENV-4). Timely and accurate diagnosis of dengue virus serotypes is crucial for the management of outbreaks. This study focussed on the development of a RT-PCR based lateral flow strip assay to detect DENV serotypes in a dual detection manner without using gel electrophoresis. The assay uses anti-biotin/streptavidin colloidal gold conjugates with fluorescent/enzymatic tagged DENV serotype specific antibodies for the direct detection of DENV infected serum samples on a nitrocellulose membrane using biotin-BSA as control line. The detection limit of the assay was up to 10 copies of cDNA for DENV-1 and 100 copies for DENV-2, DENV-3, and DENV-4. In house evaluation of DENV LFIA demonstrated 100 % sensitivity in all the serotypes compared to conventional RT-PCR, 100 % specificity for DENV-1, DENV-2, DENV-3, and 95 % specificity for DENV-4 detection. DENV serotyping was assessed in a dual detection manner (DENV-1/DENV-3 and DENV-2/DENV-4 at two test lines) on the strip. The limitation of the assay is the requirement of PCR for initial amplification and confirmation of individual serotype in case of DENV-1/DENV-3 and DENV-2/DENV-4 detection, besides the field evaluation of the assay detected DENV-2 and DENV-3 serotypes, and no other serotype was detected in line with RT-PCR findings.