Fig. 6
Amplification curves after multiplex RT-PCR from SARS-CoV-2 RNA and human RNase P transcript. (A) 10.000 c/µL SARS-CoV-2 RNA was used as template. 1 ng/µL universal human reference RNA was used to monitor amplification from the RNase P transcript. The genes nCoV_N1, N2 and E, as well as the RNase P transcript were analysed simultaneously (quadruplex RT-PCR). 120 nM purified Taq pol variants (as indicated) and 400 nM Taq pol aptamer was used for catalysis. 670 nM primer, 170 nM TaqMan probe (for nCoV_N1/N2, RNase P) and 850 nM primer, 216 nM TaqMan probe (for nCov_E) were present in the reaction mix. Reactions were monitored by measuring the fluorescence from the 6-FAM dye (nCoV_N1), the Sun dye (nCoV_N2), the Texas Red dye (nCov_E) and the Cy5 dye (RNase P). (B) SARS-CoV-2 RNA template was diluted stepwise while keeping the human reference RNA constant. The genes nCoV_N1, N2, E and RNase P transcript were analysed simultaneously (quadruplex RT-PCR). Respective average Cq values were plotted against the decadic logarithm of the template concentration. Linear regression was applied. Linear functions, R2 values and the PCR efficiencies are indicated in Supplementary Table S4. Error bars indicating the standard deviation of duplicates are shown in black. A)/B) The same mastermix was used for the control reaction (con.), but the amount of RNA template was replaced by water. Reactions were performed in duplicates.
