Table 1.
Sequences for the LDT-Quant VLCoV and LDT-Quant sgRNA assays.
Fig 1.
For every calibration run, a standard curve (shown as S1 to S6, S is for standard curve) for the Ct values obtained for 7 serial dilutions of armored RNA quantified panel (1E9 to 1E3 Copies/mL) was plotted in Prism.
Table 2.
Summary results for the specificity panel.
Table 3.
Analytical interference results.
Fig 2.
Precision of (A) LDT-Quant VLCoV and (B) LDT-Quant sgRNA assays positive extraction run controls. The precision of the High (in black) and Low (in blue) extraction controls is shown in a Levey-Jennings plot for 20 assay runs on log10-transformed values. Solid and dotted lines indicate ±3SD and ±2SD respectively.
Fig 3.
Viral load and sgRNA precision.
Three concentrations of analyte spanning the reportable range were used to evaluate the precision of the LDT-Quant VLCoV and LDT-Quant sgRNA assays. Samples at each concentration (1E6, 1E5 and 1E4 Copies/mL) were tested in duplicate in one run per day over 20 days using two operators.
Table 4.
Precision results for the LDT-Quant VLCoV.
Table 5.
Precision results for the LDT-Quant sgRNA.
Fig 4.
Plot of results from the (A) LDT-Quant VLCoV and (B) LDT-Quant sgRNA assays linearity experiment to determine reportable range. Assigned values (converted to Log10) were plotted on the x-axis versus measured values (converted to Log10) on the y-axis using EP Evaluator CLSI EP6 Linearity module based upon Log10 (Copies/mL).
Fig 5.
Probit Analysis for the (A) LDT-Quant VLCoV and (B) LDT-Quant sgRNA assays. The LLOD results were submitted to a Python script that performs a Linear Regression fit of the probit score in terms of the quantities for VL or sgRNA (in Copies/mL). The LLOD is extrapolated from the curve and as indicated by a black arrow.
Table 6.
A and B: LLOD data for the LDT-Quant VLCoV (A) and LDT-Quant sgRNA (B) assays.
Table 7.
Positive Agreement (PPA), Negative Agreement (NPA) and Cohen’s Kappa.
Fig 6.
Quantitative Method Comparison for the (A) LDT-Quant VLCoV and (B) LDT-Quant sgRNA. Results of the Manual Assay (RUO only) were compared to the Panther Fusion® LDT and plotted in EP Evaluator (Alternate Method comparison module). Deming regressions are shown below the graphs with 95% CI between parentheses.
Fig 7.
Viral trajectories of sgRNA and genomic viral loads.
Viral Load and sgRNA were quantified on the Panther Fusion® and average quantities per day (Log 10 Copies/mL) were plotted (black triangle for Viral Load and blue circle for sgRNA). The curves were generated in GraphPad with exponential growth model and indicate the dynamic tendency of viral load in analyzed samples.
Table 8.
Performance of the LDT-Quant sgRNA and LDT-Quant VLCoV using variants of concern (VOC).