A novel CRISPR-Cas12a-based diagnostic for rapid and highly sensitive detection of West Nile virus

Climate change is increasing the global threat of vector-borne diseases, including West Nile Virus (WNV), a significant human and animal pathogen transmitted primarily by <i>Culex</i> mosquitoes. Current WNV diagnostic methods, while including sensitive techniques like RT-PCR, have limitations in early detection, practicality, and cost-effectiveness. There is an urgent need to develop novel and more efficient strategies to address these challenges and to facilitate the surveillance and management of WNV infections and their spread. This study presents a highly specific and sensitive CRISPR-Cas12a-based detection protocol for WNV detection. Through systematic analysis of key reaction parameters (time: 0-60 min; reporter concentration: 1-80 nM, Cas12a and crRNA concentration: 5.625-90 nM; and template amount: 10^-2-10⁵ pg) and integration with reverse transcriptase recombinase polymerase amplification to enhance sensitivity through an isothermal technique, this assay demonstrates a novel strategy for the rapid detection of WNV, achieving 10 femtomolar sensitivity within one hour. Moreover, the assay retained its efficacy at different temperatures (25 °C and 37 °C) and in biological matrices containing the host (fly or human) genetic material, which supports its applicability in resource-limited settings. Therefore, the method presented here has the potential for broad application in diverse point-of-care settings for rapid diagnosis of WNV.