Integrated duplex reverse transcription-recombinase aided amplification (RT-RAA) and lateral flow assay (LFA) for rapid simultaneous detection of Zika virus and Japanese encephalitis virus in single reaction format.

Zika virus (ZIKV) and Japanese encephalitis virus (JEV), two consequential mosquito-borne flaviviruses, induce severe systemic and neurotropic-encephalitic pathologies with overlapping symptoms profiles, complicating differential diagnosis in co-endemic regions. To address this critical challenge, we developed a dual reverse-transcription recombinase-aided amplification (RT-RAA) coupled with duplex lateral flow assay (LFA) platform for rapid, equipment-free co-detection of ZIKV and JEV in a single reaction tube. The assay achieved isothermal amplification at 39 °C within 40 min using a field-deployable incubator. The limit of detection (LOD) reached as low as 8.5 copies ZIKV RNA, and 1.1 copies JEV RNA in single pathogen detection, while 110 copies JEV RNA in co-detection mode. This technological advance bridges the critical sensitivity gap between lab-based PCR and conventional rapid tests, enabling first-line healthcare responders to conduct precision diagnostics in non-laboratory settings from rural clinics to mobile outbreak response units.