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 in situ screening techniques to accommodate broad and large-scale testing requirements. In light of the possibility of coinfection with multiple viral strains, a multiplex detection method known as triplex reverse transcription recombinase-aided amplification (RT-RAA) has been established to facilitate the simultaneous detection of these three pathogens. The triplex real-time RT-RAA assay operates at a constant temperature of 42 degrees C and achieves detection within 30 minutes.The limits of detection (LOD) for IAV, SARS-CoV-2, and RSV were 138, 198, and 162 copies per reaction, respectively, comparable in order of magnitude to the 100 copies per reaction observed in the triplex RT-qPCR assay. Importantly, no cross-reactivity was observed with other significant viruses. In addition, compared with the triplex RT-qPCR assay, the triplex real-time RT-RAA assay demonstrated a high degree of concordance in the detection of IAV, SARS-CoV-2, and RSV in 58 clinical specimens. Overall, the triplex real-time RT-RAA assay holds considerable promise as a powerful tool for the rapid screening of respiratory diseases, owing to its advantages, including expedited detection, ease of operation, strong specificity, and high sensitivity.IMPORTANCEIt 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.