2.7 SARS-CoV-2 Omicron BA.5.2 Viral Load Reduction Assay
Viral load reduction assay was performed on VeroE6-TMPRSS2 cells.
Three-fold serially diluted mouse serum samples were preincubated with
1,000 PFU of authentic SARS-CoV-2 Omicron BA.5.2 at 37°C for 1 h.
Following preincubation, a 100 μL serum-virus mixture was added gently
to VeroE6-TMPRSS2 cells. The inoculated cells were incubated at 37℃ for
1 h. Afterwards, the infectious inoculum was aspirated, washed and
replaced with fresh medium containing the serially diluted mouse serum.
Forty-eight hours later, supernatant from the infected cells was
harvested for qRT-PCR analysis of viral load. Briefly, 50 µL of viral
supernatant were lysed with 200 µL of AVL buffer and then extracted for
total RNA using the QIAamp viral RNA mini kit (Qiagen, Germany).
Real-time one-step qRT-PCR was used for quantitation of viral load using
the QuantiNova Probe RT-PCR kit (Qiagen, Germany) with the LightCycler
480 real-time PCR system (Roche). Each 20 µL reaction mixture contained
10 µL of 2 x QuantiNova Probe RT-PCR master mix, 1.2 µL of RNase-free
water, 0.2 µL of QuantiNova Probe RT-Mix, 1.6 µL each of 10 µM forward
and reverse primer, 0.4 µL of 10 µM probe, and 5 µL of extracted RNA as
the template. Reactions were incubated at 45°C for 10 min for reverse
transcription and 95°C for 5 min for denaturation, followed by 45 cycles
of 95 °C for 5 s and 55 °C for 30 s. Signal detection and measurement
were performed in each cycle after the annealing step. The cycling
profile ended with a cooling step at 40°C for 30 s. Primers and probe
sequences were against the RNA-dependent RNA polymerase/Helicase
(RdRP/Hel) gene region of SARS-CoV-2 Omicron BA.5.2: forward primer: 5′
-CGCATACAGTCTTRCAGGCT-3′; reverse primer:
5′-GTGTGATGTTGAWATGACATGGTC-3′; specific probe: 5′-FAM
TTAAGATGTGGTGCTTGCATACGTAGAC-IABkFQ-3’.