2.1 Material
The RNA standards of RSVA, HPIV, Flu A, Flu B, and SARS-CoV-2 were purchased from Shanghai Institute of Measurement and Testing Technology. The ADV was obtained from the chicken embryo culture in our laboratory. Genomic DNA/RNA extraction kits were purchased from Tiangen Biochemical Technology (Beijing) Co. The primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The information of the respiratory virus nucleic acid is shown in Table S1. The PCR kits for nucleic acid detection of six viruses were purchased from Shenzhen Aodong Inspection and Testing Technology Co. 71 positive and 14 negative pharyngeal swab samples conserved in our laboratory were used for validation of the method.
2.2 Instruments and equipment
The Qitian RAA-B6100 thermostatic oscillation mixer was purchased from Jiangsu Qitian Genetic Biotechnology Co., Ltd., and the Genchek real-time RT-RAA fluorescence detector was purchased from Hangzhou Zhongce Bio-Sci&Tech Co. Ltd. The fluorescent quantitative PCR instrument was purchased from Bio-Rad Laboratories, Inc.
2.3 Design of primers and probes
Primers and probes were designed based on the sequence fragments in Table S2, and the corresponding sequences were downloaded from NCBI (at least 20 sequences for each virus were downloaded). Sequences were compared by DNA MAN software to obtain conserved sequences, and primers and probes were designed by custom parameters using the Primer-BLAST website and Primer 5 software, setting amplification products between 120 bp and 350 bp, primer length between 28 and 35 nucleotides, primer GC content between 20% and 70%, primer max self-complementary group was set to 4 bp and the TM value was set to 57-63°C. Several primer pairs were designed for each virus.
The probe is positioned between the forward primer (F), reverse primer (R), and is roughly 46-52 nucleotides long. The 3’ end of the probe is modified with C3 Spacer and the tetrahydrofuran (THF) in the middle of the probe serves as the cleavage site for the nucleic acid exonuclease (exo). The two T bases adjacent to the THF site are modified with a FAM fluorescent motif and a BHQ1 quenching motif, respectively. 5’ end is at least 30 nucleotides away from THF and 3’ end is at least 15 nucleotides away from THF.
2.4 Primer and probe screening
After the candidate primers and probes were identified, their relative performance had to be evaluated, compared and screened. Candidate primers and probes were screened using 105 copies/mL of template sample, and primers and probes with high fluorescence value of amplification curve, early amplification time and no non-specific amplification in negative control were selected according to the fluorescence curve detected by RAA.
2.5RT-RAA fluorescence assay.
According to the instruction of RT-RAA fluorescence amplification kit, the required amount of reaction system was prepared with the mixture solution containing enzyme-free water, A Buffer, pre-primer, forward primer, reverse primer, in Table S3. Mix all of them and add them into the assay unit tube preloaded with reaction dry powder, then add RNA/DNA template to the unit tube, and finally add 2.5 μL of B Buffer on the cap of the assay unit tube and cap the tube. Put into Qitian RAA-B6100 thermostatic oscillatory mixer to pretreat for 4 min, and put the assay unit tube into Genchek fluorescence detector and set the reaction at 39°C for 20 min.