Itaq universal probes one step rt pcr kit

The Vero E6 cells were seeded to the 24-well culture plate at 2 × 10⁵ cells/well in DMEM with 10% FBS and penicillin G sodium 100 units/ml, streptomycin sulfate 100 μg/ml, and amphotericin B 250 ng/ml (antibiotic-antimycotic, Gibco) 1 d before infection. The virus (0.02 MOI) was premixed with the test compound at indicated concentrations, solvent control, or medium containing 2% FBS (E2) for 1 hr at 37℃. Then, the mixture was added to the cells for another 1 hr of incubation at 37℃. At the end of incubation, the virus inoculum was removed and the cells were washed once with PBS buffer before adding fresh E2 medium (500 µl/well) containing test compound at indicated concentrations or solvent control for 24 hr at 37℃. Finally, the cellular RNA was extracted using NucleoSpin RNA Kit (Macherey-Nagel GmbH&Co. KG, Germany) for real-time PCR analysis of SARS-CoV-2 E genes using the iTaq Universal Probes One-Step RT-PCR Kit (Bio-Rad, USA) and the QuantStudio 5 Real-Time PCR System (Applied Biosystems, USA). All of the experiments involving the SARS-CoV-2 virus were performed in the Biosafety Level-3 Laboratory of the First Core Laboratory, National Taiwan University College of Medicine.

Vero E6 cells were seeded into 24‐well culture plates in DMEM with 10% FBS and antibiotics 1 day before infection. SARS‐CoV‐2 (multiplicity of infection, MOI = 1) was incubated with antibodies for 1 h at 37°C before adding to the cell monolayer for another hour. After removal of the virus inoculum, the cells were washed once with PBS and overlaid with 0.5 ml medium for 24 h at 37°C. The next day, the culture supernatants were harvested for RNA extraction, and the cells were retrieved for protein, RNA extraction, and immunofluorescence assays. The number of viruses in the supernatants and infected cells was determined by qPCR using the protocol provided by the WHO (https://virologie‐ccm.charite.de/en). Quantitative PCR of the E gene was performed using the iTaq™ Universal Probes One‐Step RT‐PCR Kit (172‐5140, Bio‐Rad, USA) and Applied Biosystems 7500 Real‐Time PCR software (version 7500SDS v1.5.1). A plasmid containing a partial E fragment was used as the standard to calculate the amount of viral load in the specimens. The percentage inhibition of virus yield was calculated as [1 − (Vd/Vc)] × 100%, where Vd and Vc refer to the virus copies in the presence and absence of the test compound, respectively.