Details on fluorescent probes with unique melting properties and the principles of MPA technology have been published by Fu et al. [11]. Following the manufacturer’s protocol, a single MPA reaction was performed using two PCR mixes. In tube 1, working mix 1 contained primers and probes for IFA, IFB, PIV1, PIV2, PIV3, PIV4, HADV, MP, CP, HRV and RSV, and in tube 2, working mix 2 contained primers and probes for HBOV, HMPV, COV-229E, COV-HKU1, COV-NL63, COV-OC43 and ORF1ab and N genes of SARS-CoV-2, and the design of fluorescent probes targeting these different respiratory pathogens were described in Table 1. One microliter of extracted DNA/RNA was mixed with a PCR amplification reaction mix (23 µL) containing buffer (deoxynucleoside triphosphates and Mg2). A total volume of 25 µL in each PCR tube per test was prepared by combining the master mix (Taq polymerase, UNG enzyme, and dUTP) and working mix 1 or working mix 2 (primers and probes). The PCR was run on an ABI 7500 Fast real-time PCR system (Applied Biosystems, Warrington, UK) using the following protocol: stage 1, 55 °C for 10 min, followed by 95 °C for 3 min; stage 2, 46 cycles of 95 °C for 10 s, 60 °C for 45 s and 69 °C for 20 s; stage 3, 95 °C for 10 s, followed by 25 °C for 1 min and 68°C for 15 s; stage 4, 95 °C for 10 s, 25 °C for 1 min, 68 °C for 15 s, and 60 °C for 15 s. Fluorescence measurements in the FAM, HEX/VIC, ROX and CY5 channels were recorded during stage 2 (60 °C for 45 s). The fluorescence emission raw data was continually recorded during the temperature increase procedure (stage 2), and the melting curves of FAM, VIC, ROX and Cy5 channels were generated during the dissociation stage of the PCR reaction (from 25 °C to 68 °C in stage 4). In each PCR, the internal control (CY5 detection channel) and all 18 respiratory pathogens, corresponding to the FAM (IFA, IFB and PIV3 in tube 1, SARS-CoV-2-N and SARS-CoV-2 ORF-1ab in tube 2), VIC (PIV1, PIV2, MP and CP in tube 1, COV-HKU1 and COV-NL43 in tube 2), and ROX channels (RSV, PIV4, HRV, HADV in tube 1, HBOV, HMPV, COV-229E and COV-OC43 in tube 2) were simultaneously evaluated. The fluorescent channel and melting temperature of each respiratory pathogen probe are described in Supplementary Table. Data was analyzed using the Applied Biosystems® 7500 Fast System SDS software with a single threshold to determine the quantification cycle. Samples were retested when the dye signal value of the ROX, FAM, and VIC fluorescent channels were in the gray area (35 TT value higher than 36 for cellular DNA. If any sample had a CT value less than 35 in any of the ROX, FAM, and VIC fluorescent channels, the sample was considered positive for the corresponding respiratory pathogens. External positive and negative controls are included in each run of the MPA assay to assess run validity and recognize the cross-contamination.