Simultaneous and ultrafast detection of pan-SARS-coronaviruses and influenza A/B viruses by a novel multiplex real-time RT-PCR assay

•This is the first assay to detect sarbecoviruses and flu A/B viruses in one tube.•The assay exhibits high sensitivity and specificity with LOD of 4.26–4.52 copies/reaction.•The turnround time of novel quadruplex RT-qPCR assay is 34 min.•The assay has perfect performance on both clinical and environmental samples.•The assay aids in prognose of SARS- or influenza-like cases from wild animals. Here we report an ultrafast quadruplex RT-qPCR assay with robust diagnostic ability to detect and distinguish pan-SARS-CoVs and influenza A/B viruses within 35 min. This quadruplex RT-qPCR assay comprised of one novel RNA-based internal control targeting human β2-microglobulin (B2M) for process accuracy and three newly-designed primers-probe sets targeting the envelope protein (E) of pan-SARS-CoV, matrix protein (MP) of influenza A virus and non-structural (NS) region of influenza B virus. This quadruplex assay exhibited a sensitivity comparable to its singleplex counterparts and a slightly higher to that of the Centers for Disease Control and Prevention-recommended SARS-CoV-2 and influenza A/B assays. The novel assay showed no false-positive amplifications with other common respiratory viruses, and its 95 % limits of detection for pan-SARS-CoV and influenza A/B virus was 4.26–4.52 copies/reaction. Moreover, the assay was reproducible with less than 1 % coefficient of variation and adaptable testing different clinical and environmental samples. Our ultrafast quadruplex RT-qPCR assay can serve as an attractive tool for effective differentiation of influenza A/B virus and SARS-CoV-2, but more importantly prognose the reemergence/emergence of SARS and novel coronaviruses or influenza viruses from animal spillover.