Nucleic acid extraction: Using 200 μL of virus culture supernatant, the MVR01 magnetic bead method nucleic acid extraction kit was used (article number: ZM-0044, Shanghai Zhijiang Biotechnology Co., Ltd., Shanghai, China) in an EX2400 automatic nucleic acid extraction instrument (Shanghai Zhijiang Biotechnology Co., Ltd). Virus RNA was extracted, and the elution volume was about 50 μL.

qRT-PCR: A new coronavirus nucleic acid assay kit (Cat. No. Z-RR-0479-02-50, Shanghai Zhijiang Biotechnology Co., Ltd.) was used for qRT-PCR to detect the viral load, following the manufacturer’s instructions. Briefly, the qRT-PCR amplification reaction tube was placed on a LightCycler® 480II (Roche, Basel, Switzerland) qPCR instrument, and FAM and VIC (or TEXAS RED) fluorescence channels were selected for detection. The recommended cycle parameter settings were: 45 °C for 10 min and 95 °C for 15 min; then 45 cycles of 95 °C for 15 s and 60 °C for 60 s; with single-point fluorescence detection at 60 °C. A cycle threshold (CT) value below 35 was considered effective amplification, and a CT value above 35 was considered undetected.

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