Fig. 4.

Apparent correlation plot of the rRT-PCR Ct values against the SENA mix-FCratios in SARS-CoV-2 detection. All the data of the systematic titration experiment with low concentrations of standard RNA templates (Supplementary Table 2a) and the data of clinical tests employing samples with ambiguous rRT-PCR readouts are used in this plot. In case the Ct values are too high to be detected by the rRT-PCR assay, i.e., Ct>40~45, depending on the scenarios, the mix-FCratio-correlated “apparent Ct values” may be estimated via the template concentration-related regression functions (Methods 4.5); however, majority of the extremely high “apparent Ct values” in real negative samples are arbitrary and are adopted merely to simplify the presentation. The positive cut-off of the mix-SENA detection (mix-FCratio=1.145) is defined by the C3 and C6 samples of the systematic titration experiment (Fig. 2a), while the negative cut-off of the mix-SENA detection (mix-FCratio=1.068) is defined by the P278–1 sample of the clinical tests (Supplementary Table 3). This plot confirmed the cut-off Ct values of rRT-PCR test provided by the kit supplier (BG, ref to all detectable Ct values shown as solid dots). Meanwhile, with the aid of mix-SENA, the sensitivity of rRT-PCR was increased up to the detected level of O-Ct=43.3 in samples of D2 and D4 and estimated level of O-Ct=49.5 in samples of C6 and P280. In addition, false positives were detected with O-Ct values as low as 39 (P278–1), which was also the key test to define the negative cut-off of mix-SENA.