Abstract
The diagnosis of Dengue and Chikungunya infections during acute phase is a priority considering emerging pattern and increasing trends of their infections. The present study describes the commercial development and validation of RT-PCR test for the simultaneous detection of of DEN and CHIK viral RNA in a single tube from human plasma samples. Multistep one step RT-PCR assay was developed and validated for detection and discrimination of DEN and CHIK along with exogenous internal control. The test was evaluated for commercial use using 3 different lots to determine analytical sensitivity, specificity, precision and stability. The external clinical evaluation was performed at NABL accredited lab with known positive and negative Chikungunya and Dengue specimens and comparator assay method. The findings showed that the test could identify CHIK and DEN viral nucleic acid in clinical samples within 80 min, without any cross-reactivity. The analytical detection limit of the test was 1.56 copies/µl for both. The clinical sensitivity and specificity was ≥ 98% and provide a high-throughput and screen up to 90 samples in a single run. It is available in a freeze-dried format and can be used in both the manual and automated platforms. This unique combo test, PathoDetect™ "CHIK DEN Multiplex PCR Kit" enables simultaneous, sensitive, specific detection of DENV and CHIKV and serves as "ready to use" platform for commercial use. It would aid the differential diagnosis as early as day 1 of the infection and facilitate screen-and-treat approach.