Multiplex real-time reverse transcription recombinase-aided amplification assay for the detection of SARS-CoV-2, influenza A virus, and respiratory syncytial virus.

Influenza A virus (IAV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and respiratory syncytial virus (RSV) are significant pathogens that induce respiratory symptoms in humans, significantly impacting healthcare systems, economic activities, and human life on a global scale. The clinical manifestations associated with SARS-CoV-2, IAV, and RSV exhibit similarities. Cases of clinical coinfection are prevalent, highlighting the necessity for rapid differential diagnosis to avoid treatment delays. Traditional reverse transcription quantitative polymerase chain reaction (RT-qPCR) methods are often time-consuming and limited to central laboratories, necessitating the development of rapid and IMPORTANCE: It is well established that SARS-CoV-2, IAV, and RSV are significant pathogens that elicit respiratory symptoms in humans. In this study, we successfully developed a triplex real-time RT-RAA assay for the simultaneous rapid detection of SARS-CoV-2, IAV, and RSV. This assay is distinguished by its high specificity, sensitivity, and user-friendliness. It addresses the limitations associated with the necessity of specialized biological laboratories and the time-consuming, complex procedures that impede rapid diagnosis. This assay holds significant importance for the early diagnosis and epidemiological surveillance of respiratory tract infections, providing comprehensive and timely diagnostic information for clinical practice. Furthermore, this assay serves as a valuable tool for facilitating extensive and large-scale screening of SARS-CoV-2, IAV, and RSV.