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.