Diagnostics
Development of a Single Thermal Cycling Multiplex RT-qPCR Assay for Rapid Arbovirus Detection
By Róger J. Costa, Andreas Stöcker, Cleyde Sheyla Chachaqui Marconi and 6 others.
Abstract
Background
The co-circulation of multiple arboviruses with similar clinical presentations poses a significant challenge for public health surveillance. This study aimed to develop and clinically validate a novel multiplex RT-qPCR assay for the simultaneous detection of five arboviruses in three reactions.
Methods
A diagnostic accuracy study was conducted in Salvador, Brazil. Evaluation was performed using 254 prospective serum samples and 34 archived positive samples. The primers and probes for the assay were designed in silico, all targeting highly conserved regions of Dengue (DENV), Zika (ZIKV), Chikungunya (CHIKV), Oropouche (OROV), and West Nile (WNV) viruses. The protocol was optimized in three reactions with a single thermal cycle, comprising a tetraplex reaction (DENV, CHIKV, ZIKV, and hRNAseP, an endogenous control) and two singleplex reactions (OROV and WNV).