1. Cells, instruments, reagents, viruses
VERO-E6 cells are stored in the laboratory of Zhejiang University. DMEM medium, FBS, P/S, and PBS buffer are GIBCO products. Methylene blue is a product of Jichuan Pharmaceutical Group Co., Ltd. Human plasma is donated by volunteers. All experimental operations involving live viruses were carried out in Zhejiang University's Biosafety Tertiary Laboratory (BSL-3). The research protocol was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University Medical College.
2. Methylene blue light inactivated virus
To 180 mL of healthy human plasma, 20 mL of indicator virus was added at a 9: 1 (V: V) ratio, and mixed. 1 methylene blue (MB) injection for clinical use (20mg / 2mL / branch) was diluted 13.4 times with normal saline to make up a 2mM / L methylene blue volume solution, and then diluted to 2000, 1000 and 500 times. This mixture was added to 200 mL of plasma virus mixture and mixed to a final concentration of 1 uM, 2 uM, or 4 uM. "BX-1 AIDS treatment instrument" was used at room temperature, illumination adjusted to 55,000 ± 0.5 million Lux, and irradiated 0, 2, 5, 10, 20, 40 mins under single wavelength of 630nm light. About 1 mL of plasma was taken at each time point, diluted 10-fold with DMEM medium containing 2% FBS, and then filtered through a 0.45 μm filter for virus titer detection. At the same time, the virus control was set (only virus in the plasma, left at room temperature for 40 mins as an untreated control), and control with pure methylene blue (MB) was set (virus and methylene blue 4 μM added to the plasma, with no light treatment, and allowed to stand for 40 mins as a control for the effect of methylene blue on the virus) and a finally the light-only control was set (only the virus was added to the plasma, and light was used for 40 mins as a control of the effect of light on virus).
3. Virus titer measurement
After trypsinization of VERO cells, 1 × 104 cells/well were inoculated into 96-well cell culture plates at 100ul of culture medium per well. After the cells grew into a single layer in a 96-well plate, the culture medium was discarded and the treated plasma was seeded. The virus was log-diluted with 2% FBS in DMEM medium from 10-2-10-7. This process was repeated for 4 wells per dilution, 200ul/well. Normal cell control wells were established (with cells, virus-free). The 96-well plate was placed in a 5% CO2 and 37 ° C incubator. After 3 h of incubation, the supernatant was washed off, and 200 uL/well of 2% FBS DMEM medium was added. Cell lesions were observed every 24 h until 6 d. TCID50 was calculated according to the Reed-Muench method. The cell culture supernatant at 6 d of the culture was pipetted, and the viral nucleic acid load was measured.
4. Three Generations of Blind Tests
1 mL of the test group with virus and 1,2,4 uM methylene blue was irradiated for 40 min. It was then diluted 10 times with 2% FBS DMEM medium, filtered with 0.45 μm filter (Millipore), and added to VERO cells every 48 h. 1 mL of the supernatant was diluted 10 times with 2% FBS DMEM medium, and added to VERO cells for 3 passages. These were observed for cytopathic effects. For the duration of the 3rd generation of cell blind transmission, cytopathic lesions were positive (+) at any time, indicating that the virus was not completely inactivated; negative (-), indicating that the virus had been completely inactivated.
5. Viral load of culture supernatant measured by qRT-PCR
Nucleic acid extraction: with 200uL of virus culture supernatant, the MVR01 magnetic bead method nucleic acid extraction kit was used (article number: ZM-0044, Shanghai Zhijiang Biotechnology Co., Ltd.) in the EX2400 automatic nucleic acid extraction instrument (Shanghai Zhijiang Biotechnology Co., Ltd. Virus nucleic acid 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 viral load. The specific steps were as follows: tn × 19 uL of new coronavirus (2019-nCoV) nucleic acid fluorescent PCR detection mixture and n × 1 uLRT-PCR enzyme (n is the number of reaction tubes) were shaked and mixed for several seconds, and centrifuged at 3000 rpm for several seconds. 20 uL of the above mixture was placed into a PCR tube, to which was added 5 uL each of the sample nucleic acid extraction solution, DEPC-H2O, and positive control to the PCR tube. The tube cap was covered and the PCR amplification reaction was immediately performed. The PCR amplification reaction tube was placed on a LightCycler® 480II (Roche) real-time quantitative PCR instrument, and FAM and VIC (or TEXAS RED) fluorescence channels were selected for detection. Recommended cycle parameter settings: 45 ℃ × 10min; 95 ℃ × 15min; then 95 ℃ × 15sec, 60 ℃ × 60sec, cycle 45 times; single-point fluorescence detection at 60 ℃. A CT value below 35 was considered effective amplification, and a CT value above 35 was considered undetected.