Table 1. Analytical sensitivity. The limit of detection of the 2019-nCoV TaqMan RT-PCR Kit Dx was determined using 20 contrived samples of each sample type. Contrived nasopharyngeal swabs (A), oropharyngeal swabs (B) and saliva samples (C) were generated by spiking 2019-nCoV RT-PCR Positive Control corresponding to 10 copies per PCR reaction. Confirmatory results were acceptable at a 95% confidence interval. This can be achieved when obtaining a minimum of 19 positive samples out of the 20 samples spiked at the limit of detection. As seen in Table 1, the limit of detection of Norgens 2019-nCoV TaqMan RT-PCR Kit Dx from RNA isolated from nasopharyngeal swabs, oropharyngeal swabs and saliva samples was confirmed to be 10 copies per PCR reaction at a 95% confidence interval.

Table 2: Specificity analysis To test the analytical specificity of Norgens 2019-nCoV TaqMan RT-PCR Kit Dx, the 2019-nCoV RT-PCR Positive Control Dx (containing the two nCoV nucleocapsid target gene RNA (N1 and N2) and RNase P) was used to test the kits specificity against RNA purified from other related pathogens.   As can be seen in the table only Norgens 2019-nCoV RT-PCR Positive Control showed amplification for all genes. Therefore, Norgens 2019-nCoV TaqMan RT-PCR Kit Dx can be used to specifically detect, confirm and discriminate COVID-19.

Table 3: Diagnostic Accuracy. Clinical evaluation of the accuracy of the 2019-nCoV TaqMan RT-PCR Kit Dx was conducted with contrived nasopharyngeal swabs, oropharyngeal swabs and saliva samples by testing 30 positive and 30 negative samples to generate the Positive Percentage Agreement (PPA), Negative Percentage Agreement (NPA) and overall percentage agreement (OPA) as a measurement of estimated Diagnostic Accuracy. For the 30 contrived positive samples, each were spiked with 5 µL of different concentrations of the 2019-nCoV RT-PCR Positive Control to generate input  15 samples of variable transcript content that corresponds to a limit of detection (LoD) range from 1X to 1,000X (10 samples at 1X, 10 samples at 2X, 4 samples at 10X, 3 samples at 100X and 3 samples at 1,000X). The remaining 30 samples from each sample type were not spiked (nonreactive). RNA isolation was performed from all samples using Norgen’s Saliva/Swab RNA Purification Kit (Cat. #69100) and RNA was eluted in 50 µL. Five microliters of the isolated RNA were used as a template in the Norgen’s 2019-nCoV TaqMan RT-PCR Kit Dx to detect the 3 targets of the kits (N1, N2 and RP). The various SARS-CoV-2 kit targets can be detected from RNA isolated from contrived nasopharyngeal swabs, oropharyngeal swabs and saliva samples, at various detection limits using Norgen’s 2019-nCoV TaqMan RT-PCR Kit Dx with no detectable viral targets from non-reactive samples.

Table 4. Interpretation of Assay Results. The Negative Control (NTC No Template Control) reaction(s) must be negative and not exhibit fluorescence growth curves that cross the threshold line. If there is any amplification with the NTC the run is not valid and no interpretation of 2019-nCoV detection can be made. In this case the assay must be repeated. The Positive Control (PosC) reaction(s) should produce a positive result with an expected Ct value below 20 for each target. If the positive control does not provide a positive result the run is not valid and no interpretation of 2019-nCoV detection can be made and the assay must be repeated. If the NTC and PosC are exhibiting the correct results, the results of the detection assays can be interpreted as outlined in the product manual.