3. Results
3.1 Primers and fluorescent probes screening
We downloaded 20 sequences of each virus from NCBI for alignment and obtained the relatively conserved sequences of each virus, and the conserved sequences are shown in Table S2. According to the RT-RAA design principles, the appropriate probes were first designed in the selected conserved sequences of each virus, as shown in Figure 3, and the probe positions of each virus were P (132-179) in RSV gene sequence, P (108-156) in HPIV gene sequence, P (714-763) in Flu A gene sequence, P (557-603) in Flu B gene sequence probe sequence, P (330-377) in SARS-CoV-2 gene sequence, and P (412-474) in ADV gene sequence. Then, several forward and reverse primers were designed surrounding to the probe, while some primers that might form secondary structures by themselves were excluded, and the relative positions of the candidate primers are shown in Figure 3. The ideal primers were screened by RT-RAA experiments based on the fluorescence intensity and peak onset time of each group of designed primers (Figure S1). The sequence information of the ideal primer pairs is shown in Table 1.