Qiaamp viral kit

We identified all of the collected clinical specimens of ILI and SARI using the centres for Disease Control (CDC) (Atlanta, United States) real-time reverse transcription polymerase chain reaction (RT-PCR) typing and sub-typing assay.¹¹ This assay is based on TaqMan chemistry and includes a panel of oligonucleotide primers and dual-labelled hydrolysis probe sets for universal influenza A and B, H1pdm09, H3, H5, and RNP (ribonucleoprotein). Viral ribonucleic acid (RNA) was extracted from 140 µL of the nasopharyngeal specimens using a QIAamp Viral Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The amplification was performed using the SuperScript™ III Platinum Taq one-step quantitative kit (Invitrogen, Carlsbad, CA, USA) in the IQ5 quantitative PCR system (Bio-Rad, Hercules, CA, USA). The total volume of the amplification reaction was 25 μL, consisting of 12.5 mL of 2× buffer, 0.5 μL of enzyme mix, 0.5 μL of both forward and reverse primers (40 mM), and 0.5 μL of probe (10 mM) and Diethylpyrocarbonate (DEPC)-treated water each, which added to a total volume of 20 μL. Finally, 5 μL of viral RNA extracted from the clinical samples was added to the real-time RT-PCR assay mix.

The functional test of the condensation fluid was done by comparing its ability to block SARS-CoV-2 entry into Vero-E6 cells between pre-nebulized rACE2 and condensation fluid. A total of 5 × 10⁴ Vero-E6 cells were seeded per well in a 48-well culture plate in DMEM containing 10% FBS 24 h before infection with SARS-CoV-2 or with mixes containing SARS-CoV-2 + rACE2. The stocked rACE2 and condensation fluid containing nebulized rACE2 at indicated concentrations were mixed with SARS-CoV-2 at MOI 2.0 in a final volume of 100 μL per well in DMEM (0% FBS) for 30 min on ice and then added to Vero-E6 cells (the inoculum remained unremoved). At 15 h post-infection, the supernatants were removed, and the cells were washed three times with PBS and then lysed using Buffer AVL (QiaAmp Viral kit, Qiagen) for RNA extraction for viral envelope (E) gene target assay by qRT-PCR.