2019 ncov cdc ddpcr triplex probe assay

SARS-CoV-2 viral load was quantified by reverse transcriptase droplet digital PCR (RT-ddPCR), including the one-step reverse transcription (One-Step RT-ddPCR Advanced Kit for Probes, Bio-Rad Laboratories, Hercules, CA, USA) and the triplex probe assay for PCR amplification (2019-nCoV CDC ddPCR Triplex Probe Assay, Bio-Rad Laboratories, Hercules, CA, USA). The assay contains primers and probes targeting two regions of the SARS-CoV-2 nucleocapsid gene (N1 and N2) and the human Rnase P gene (RPP30). The reaction mixture was performed with 5.5 µL of SARS-CoV-2 RNA sample and following the manufacturer’s instructions. All the samples were tested in duplicate. Data analysis was performed using the QuantaSoft Analysis Pro Software (v. 1.0.596, Bio-Rad Laboratories, Hercules, CA, USA), which showed the results as copies per microliter of 1x ddPCR reaction. All viral load values were recalculated to copies per milliliter of swab, and the sensitivity threshold was 100 copies/mL. To assess the accuracy of the absolute viral RNA quantification, two fold serial dilutions of the positive control were analyzed for lineal regression analysis. Finally, to establish a reliable comparison between the viral load of different samples, the absolute quantification of SARS-CoV-2 RNA was normalized with RPP30 and expressed as copies/10⁴ cells (Figure S1).

Viral nucleic acid was extracted from 200 μL of heat-inactivated SARS-CoV-2 stock using the chemagic MSM I system (PerkinElmer, Santa Clara, CA) and eluted in a total volume of 80 μL. ddPCR was performed using the one-step RT-ddPCR advanced kit for probes (Bio-Rad, Hercules, CA) with the 2019-nCoV CDC ddPCR triplex probe assay (catalog number dEXS28563542; Bio-Rad) targeting the N1 and N2 genes, following the manufacturer’s protocol. Briefly, droplet generation was performed with the AutoDG system (Bio-Rad), and amplification was performed with a C1000 thermocycler (Bio-Rad); plates were read with a QX200 droplet reader (Bio-Rad), and results were analyzed manually using QuantaSoft Analysis Pro v1.0.596 (Bio-Rad).

Viral nucleic acid was extracted from 200 μL of heat-inactivated SARS-CoV-2 stock using the chemagic MSM I system (PerkinElmer, Santa Clara, CA) and eluted in a total volume of 80 μL. ddPCR was performed using the one-step RT-ddPCR advanced kit for probes (Bio-Rad, Hercules, CA) with the 2019-nCoV CDC ddPCR triplex probe assay (catalog number dEXS28563542; Bio-Rad) targeting the N1 and N2 genes, following the manufacturer’s protocol. Briefly, droplet generation was performed with the AutoDG system (Bio-Rad), and amplification was performed with a C1000 thermocycler (Bio-Rad); plates were read with a QX200 droplet reader (Bio-Rad), and results were analyzed manually using QuantaSoft Analysis Pro v1.0.596 (Bio-Rad).