Quantification of the Inhibition in the Six-plex Digital PCR Assay targeting SARS-CoV-2 (N1 and N2), MHV, IAV, IBV, and RSV v1

This protocol describes the procedure for determining PCR inhibition in the RESP6 digital PCR assay, using SARS-N1 as a representative target. The assay was developed within the context of Pitton et al. (2024) DOI: 10.1038/s44221-025-00503-x. To assess inhibition, a known quantity of synthetic SARS-N1 RNA is spiked into the dPCR mastermix. The inhibition calculation entails a two-run approach: first, SARS-N1 is quantified using an unspiked mastermix on the RESP6, followed by a second run with a spiked mastermix as described in this protocol. The percentage of signal lost because of PCR inhibition is calculated using the following equation: 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 (%) = (1 − (𝑆𝐴𝑅𝑆𝑁1 𝑖𝑛 𝑠𝑝𝑖𝑘𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒/(𝑆𝐴𝑅𝑆𝑁1 𝑖𝑛 𝑢𝑛𝑠𝑝𝑖𝑘𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒 + 𝑆𝐴𝑅𝑆𝑁1 𝑖𝑛 𝑠𝑝𝑖𝑘𝑒𝑑 𝑁𝑇𝐶))) ∗ 100%