SARS-CoV-2 Utility Channel test performance evaluation
Limit of detection (LoD), inter-run variability and cross-reactivity with other respiratory pathogens were determined. In-vitro transcribed RNA (IVT RNA) of the E gene of SARS-CoV-2 and purified RNA of SARS-CoV (strain Frankfurt-1) were used as positive controls (obtained via the European virus archive global (EVAg), https://www.european-virus-archive.com) [7]. Limit of detection of the SARS-CoV-2 UCT was determined by analysing each of eight replicates of a dilution series containing IVT RNA diluted in E-swab medium (Copan, Brescia, Italy; modified liquid Amies medium) and Roche cobas PCR medium (1:1) at 10,000, 1,000, 500, 250 and 125 copies/mL and eight negative samples. The LoD was 689.3 copies/mL with 275.72 copies per reaction at 95% detection probability (Figure 1, Figure 2). For estimation of inter/intra-run variability, we analysed each of two concentrations (ca 5 × and 10 × LoD spiked IVT SARS-CoV-2 RNA) in five replicates and a negative sample with five replicates in two runs each. Minimal deviation was observed with ± 0.5 cycle threshold (Ct) at 10 × LoD and 0.75 Ct at 5 × LoD. No false positive results occurred.
Figure 1  Example for amplification curves of the SARS-CoV-2-UCT SARS-CoV-2 Utility Channel test
IVT RNA: in-vitro transcribed RNA.
Dark blue curves: specific SARS-CoV-2 signals of IVT-RNA at 100,000 copies/mL (1), 10,000 copies/ml (2), 1,000 copies/ml (3) and 100 copies/ml (4). Light blue curves: signals of the internal control (5).
Figure 2  Determination of limit of detection of the SARS-CoV-2 Utility Channel test based on in-vitro transcribed RNA
IVT RNA: in-vitro transcribed RNA.
Probit analysis revealed a limit of detection of 689.3 copies/mL at 95% probability. Blue line: probit curve (dose-response rule); red dotted lines: 95% confidence intervals. IVT-RNA obtained by EVAg. Potential interference of the SARS-CoV-2 UCT with other respiratory pathogens (including other human CoV strains) was evaluated by analysing 88 previously determined clinical samples and an external quality control assessment panel (INSTAND, Düsseldorf, Germany) containing lysates of infected cells. None of these organisms were detected by the SARS-CoV-2 UCT assay (Table 2), confirming high specificity of the assay for viruses within the Betacoronavirus subgenus Sarbecovirus [4].
Table 2  Potential cross-reactivity SARS-CoV-2 Utility Channel test with other respiratory pathogens, evaluated with a panel of organisms typically found in respiratory infections
Clinical samples with known viruses  Number tested
Coronavirus (not typed)  5
hCoV HKU-1  2
hCoV NL63  1
Adenovirus  2
Bocavirus  7
Human metapneumovirus  6
Influenza A  7
Influenza A(H1N1)  6
Influenza B  3
Parainfluenza 1 virus  3
Parainfluenza 2 virus  1
Parainfluenza 3 virus  8
Parainfluenza 4 virus  3
Respiratory syncytial virus (A/B)  10
Rhino/enterovirus  8
Chlamydophila pneumoniae  4
Mycoplasma pneumoniae  4
Legionella pneumophila  3
Bordetella pertussis  4
Bordetella parapertussis  1
Total number of clinical samples   88
External quality control assessment panel with known viruses (lysates of infected cells)
hCoV 229E  1
hCoV NL63  1
hCoV OC43  3
MERS-CoV  4
Coxsackievirus A21  1
Coxsackievirus B3  1
Enterovirus 68  1
Rhinovirus  3
Human metapneumovirus  3
Virus-negative  4
Total number of quality control assessment panel samples   22
Total number of samples   110
hCoV: human coronavirus;
Clinical samples had been pre-analysed by routine diagnostic. External quality control assessment panel (INSTAND) samples contained inactivated lysates of virus infected cells including four virus-negative cell lysates.