2.3.2. Droplet Digital PCR (ddPCR)

For the detection of SARS-CoV-2, the One-Step RT-ddPCR Advanced Kit for Probes (BioRad, Richmond, CA) was used. For each 20 μl reaction, 5 μl of Supermix, 2 μl of Reverse transcriptase, 1 μl of 300 mM DTT, 1 μl of Probe (1:40), 1 μl of both Forward and Reverse primers (1:10) and 4 μl of H2O were provided and 5 μl of starting RNA was added. The oligonucleotides and probes used in RT‐ddPCR were the same used in RT‐qPCR. For the droplet generation in RT‐ddPCR, 20 μl of reaction volume was transferred to 8‐channel disposable droplet generation cartridge and 70 μl of droplet generation oil for probe was added in adjacent oil wells. The cartridge was placed into a QX200 droplet generator (BioRad, Richmond, CA), which makes the partition of each sample into droplets. The droplets were carefully transferred to a semi‐skirted 96‐well PCR plate (BioRad, Richmond, CA), sealed using the PX1 PCR Plate Sealer (BioRad, Richmond, CA) for PCR in 2720 Thermal Cycler (Applied Biosystems, USA). After PCR of targets presented in the droplets, the QX200 droplet reader (BioRad, Richmond, CA) was used to analyze each droplet individually, which counts positive and negative droplets to establish absolute quantification of samples (concentration).

The QuantaSoft 1.6 software was used to view fluorescence data in 1D amplitude, concentration data, copy number data, events (number of positive, negative or total droplet counts). The multi‐well threshold tool was used in all the wells according to results of specificity assay in negative samples to discriminate between positive and negative droplets. The software automatically reported the copy number of each sample.