Virus strains used in this study.

Chikungunya virus (CHIKV) is a positive sense RNA Alphavirus that continues to pose major public health threats throughout the world. CHIKV is primarily transmitted via the Aedes genus mosquito; however, has also exhibited transmission routes via blood transfusion and vertical transmission (mother to child). With only one approved vaccine thus far and no approved medicines or specific therapeutics, early detection is crucial in mitigating potential CHIKV outbreaks. Here, we designed and evaluated a sensitive and specific CHIKV diagnostic using reverse transcription-recombinase aided amplification (RT-RAA) coupled lateral flow strip detection (LFD) targeting a highly conserved region of the CHIKV E1 gene. Our results demonstrate that using our simple sample preparation reagent (TNA-Cifer-E), we can inactivate live CHIKV in two minutes at room temperature, whilst also sustaining viable viral RNA. Our specificity analysis demonstrates the Iso-CHIKV-Dx does not detect any closely related Alphaviruses nor any of the common co-circulating Flaviviruses. Proof-of-concept evaluation using urine spiked with CHIKV exhibited that in CHIKV infected urine samples, our Iso-CHIKV-Dx can detect as low as 570 copies/µL of CHIKV RNA in 30 minutes under isothermal conditions. Contrary to conventional RT-qPCR, our Iso-CHIKV-Dx does not require expensive machinery, advanced instrumentation or extensively trained personnel. Further performance comparisons also show that our Iso-CHIKV-Dx is four times faster than conventional RNA isolation and RT-qPCR. As such, pre-clinical, proof-of-concept evaluation demonstrates that our Iso-CHIKV-Dx has the potential to act as a robust, point of care CHIKV diagnostic that could prove to be highly beneficial in place of, or in the absence of conventional diagnostic approaches such as RT-qPCR.