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.