SARS-CoV-2 nucleic acid expression was detected with SARS-CoV-2 virus kits via quantitative reverse transcription polymerase chain reaction (2019-nCoV Nucleic acid detection Kit, Daan Gene, Guangzhou, China) according to the national guideline and the manufacturer’s instructions, and all patients were examined daily during hospitalization. The conditions for SARS-CoV-2 nucleic acid amplification were 50 °C for 15 min, 95 °C for 15 min, followed by 45 cycles of 94 °C for 15 s and 55 °C for 45 s. Positive detection was defined as a cycle threshold (Ct value) less than 40(500 copies/ml), and the test procedure was carried out in strict accordance with the protocol. A patient’s viral load was defined as the patient’s lowest Ct value during hospitalization, and the time to peak viral load was defined as the time from the first positive SARS-CoV-2 test to the lowest Ct value during hospitalization. The incubation period was defined as the number of days from contact exposure to the onset of a positive nucleic acid result.
Serum antibodies (anti-Spike IgG and IgM) against SARS-CoV-2 were detected via a commercial ELISA kit (SARS-CoV-2 Ig G and SARS-CoV-2 IgM, Maccura Biotechnology Co., Ltd., Chengdu, China) according to the manufacturer’s instructions, and the antibodies were detected at admission, day 7, day 10, day 14 and at discharge. Briefly, 96-well plates were coated with purified SARS-CoV-2 antigen in phosphate buffer overnight at 4 °C in physiological saline (PBS). We added 10 µl of each serum sample to a reaction plate, mixed it with 50 µl magnetic beads and 50 µl buffer, incubated it in the reaction plate for 10 min and then washed it. Then, we added 100 µl acridine ester-labeled recombinant magnetic beads and incubated them in the reaction plate for 10 min. After washing, we added the substrate solution and mixed it well to detect the luminescence signal value. The assay detects antibodies to the spike protein of SARS-CoV-2 with a cut-off (CO) value of <1.0 for negative results and the value ≥1.0 were defined as positive, the final value of the test was calculated with the optical density value of the sample/CO value.
Serum antibodies (anti-Spike IgG and IgM) against SARS-CoV-2 were detected via a commercial ELISA kit (SARS-CoV-2 Ig G and SARS-CoV-2 IgM, Maccura Biotechnology Co., Ltd., Chengdu, China) according to the manufacturer’s instructions, and the antibodies were detected at admission, day 7, day 10, day 14 and at discharge. Briefly, 96-well plates were coated with purified SARS-CoV-2 antigen in phosphate buffer overnight at 4 °C in physiological saline (PBS). We added 10 µl of each serum sample to a reaction plate, mixed it with 50 µl magnetic beads and 50 µl buffer, incubated it in the reaction plate for 10 min and then washed it. Then, we added 100 µl acridine ester-labeled recombinant magnetic beads and incubated them in the reaction plate for 10 min. After washing, we added the substrate solution and mixed it well to detect the luminescence signal value. The assay detects antibodies to the spike protein of SARS-CoV-2 with a cut-off (CO) value of <1.0 for negative results and the value ≥1.0 were defined as positive, the final value of the test was calculated with the optical density value of the sample/CO value.
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