One-step RT-dPCR for SARS-CoV-2 N gene quantification

After RNA extraction with the Qiagen QiaAmp RNA kit, one step RT-dPCR reactions were carried out as described in [9 (link)]. For N gene quantification, a duplex assay was performed using China N and SarE genes with primers and probe concentrations of 400 nM and 200 nM, respectively, and optimized by varying the annealing temperature. The sequence of China N and SarE primers is described in [9 (link)–11 (link)], Droplet generation was conducted as described in [9 (link)]. The PCR was performed under the following conditions: 60 min reverse transcription at 50 ºC and 10 min enzyme inactivation at 95 ºC followed by 45 cycles using a two-step thermal profile of 30 s denaturation at 95 ºC and 60 s annealing and extension at 55 ºC; followed by 10 min at 98 ºC and then cooled to 4 ºC. Following thermal cycling, the PCR plates were transferred to a droplet reader (QX200 BioRad, USA) and the data analyzed using QuantaSoft Analysis Pro 1.0.596 (BioRad, USA).