Shifting the paradigm in RNA virus detection: integrating nucleic acid testing and immunoassays through single-molecule digital ELISA.

In this review article, we explore the characteristics of RNA viruses and their potential threats to humanity. We also provide a brief overview of the primary contemporary techniques used for the early detection of such viruses. After thoroughly analyzing the strengths and limitations of these methods, we highlight the importance of integrating nucleic acid testing with immunological assays in RNA virus detection. Although notable methodological differences between nucleic acid testing and immune assays pose challenges, the emerging single-molecule immunoassay-digital ELISA may be applied to technically integrate these techniques. We emphasize that the greatest value of digital ELISA is its extensive compatibility, which creates numerous opportunities for real-time, large-scale testing of RNA viruses. Furthermore, we describe the possible developmental trends of digital ELISA in various aspects, such as reaction carriers, identification elements, signal amplification, and data reading, thus revealing the remarkable potential of single-molecule digital ELISA in future RNA virus detection.