Figure 1

Design of chimeric controls for SARS-CoV-2. (a) View of the SARS-CoV-2 genome showing genes targeted by WHO-published real-time RT-PCR primer/probe sets (ORF1a, ORF1b, E and N genes). Specific binding sites targeted by different countries are shown in individual tracks (CN = China, DE = Germany, FR = France, HK = Hong Kong, TH = Thailand and US = United States). Targeted regions encompassing several primer/probe sets were selected and exclusively partitioned between chimeric A/B standards. The expected amplicons for the CDC test are shown in the last track. (b) The different targeted regions for standards A and B were shuffled and joined together to form chimeric sequences. The paired design of chimeric A/B standards, where a target in A is absent in B (and vice versa), enables the synthetic RNA transcripts to simultaneously act as positive and negative controls for the real-time RT-PCR primer/probe sets (F = forward primer, R = reverse primer and P = probe). This enables internal cross validation of positive and negative controls between standards A and B. The vector backbone was omitted from the representation of the chimeric A/B standards. The colours for targeted regions in the chimeric A/B standards are the same between figures (a) and (b).