SARS-CoV-2 Utility Channel test setup
A custom-made primer/probe set based on a previously published assay targeting the E gene [4] was optimised for the use on the automated system. Primer and probes were ordered from IDT DNA Technologies (Coralville, United States (US)). Both primers were modified with 2’-O-methyl bases in their penultimate base to prevent formation of primer dimers. The ZEN double-quenched probe (IDT) was used in order to lower background fluorescence. The master mix (Mmx) cassette (for 96 tests) is prepared by combining 84 µL forward primer (400 nM, 5´-ACAGGTACGTTAATAGTTAATAGCmGT-3´), 84 µL reverse primer (400 nM, 5´-ATATTGCAGCAGTACGCACAmCA-3´) and 10.5 µL probe (50 nM, 5´-Fam-ACACTAGCC/ZEN/ATCCTTACTGCGCTTCG-Iowa Black FQ-3’)* together with 182 µL water and 5,640 µl Mmx2 mixture. After mixing, the 6 mL are transferred to the reagent cassette. The Mmx cassette is delivered with a full-process control: it is preloaded with an internal control (IC) RNA together with primers and probe of the IC detection assay by default [6]. Instrument settings in the cobas mni Utility Channel software (Roche, Los Gatos, US) and the temperature profile used for the RT-PCR reaction are summarised in Table 1.
Table 1  SARS-CoV-2 PCR cycling conditions and software settings in the Utility Channel software used to create the run template
Sample type  Alcohol-based swab (400 µL input)
Channels  1  2:SARS-CoV-2 E-gene  3  4  IC
RFI  NA  1.5  NA  NA  Predefined
PCR cycling conditions  UNG incubation  Pre-PCR step  1st measurement  2nd measurement  Cooling
Number of cycles  Predefined  1  5  45  Predefined
Number of steps  3  2  2
Temperature  55 °C; 60 °C; 65 °C  95 °C; 55 °C  91 °C; 58 °C
Hold time  120 s; 360 s; 240 s  5 s; 30 s  5 s; 25 s
Data acquisition  None  End of each cycle  End of each cycle
IC: internal control; NA: not applicable; RFI: relative fluorescence increase; UNG: uracil-DNA N-glycosylase. Assay performance was evaluated for swab samples. All clinical specimens used were collected at the University Medical Center Hamburg-Eppendorf (UKE-HH). Samples were mixed 1:1 with Roche cobas PCR media (≤ 40% guanidine hydrochloride in Tris-HCL buffer) and incubated for 30 min before loading onto the cobas 6800 system. Apart from that sample preparation step, no further manual steps are required during the entire workflow of the novel assay.