A Rapid RT-RAA Assay for Visual Detection of Ebola Virus: Advancing Early Diagnosis in Resource-Limited Settings.

Ebola virus (EBOV) infection constitutes a significant global public health threat, and no curative treatment is currently available for it. Rapid and accurate detection of EBOV nucleic acid is crucial for controlling the spread of Ebola virus disease (EVD). The gold standard for EBOV diagnosis is real-time reverse transcription polymerase chain reaction (RT-qPCR), which requires costly equipment and skilled personnel, potentially hindering its application for rapid detection, especially in resource-limited settings. Consequently, there is an urgent need to develop a simple, accurate, and rapid diagnostic method for EVD. In this study, a real-time reverse transcription recombinase-aided amplification (RT-RAA) assay was developed for the specific visual detection of the conserved region of the EBOV nucleoprotein (NP) gene. The RT-RAA assay can be completed within 30 min at 42 °C, and results can be visualized using a portable blue light imager. The assay exhibited strong analytical specificity toward EBOV. No cross-reactivity was observed with any of the other public-health-relevant viruses tested. The visual RT-RAA assay demonstrated sensitivity comparable to RT-qPCR, detecting 52 copies per reaction at a 95% probability level, whereas RT-qPCR required 74 copies per reaction. The RAA method demonstrated excellent repeatability and stability, with intra-assay and inter-assay CVs less than 5% and 7%, respectively. These results clearly indicate that the visual RT-RAA method is specific, accurate, simple, rapid, and reliable for EBOV detection.