PMC:7068162 / 2048-4432 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7068162","sourcedb":"PMC","sourceid":"7068162","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7068162","text":"SARS-CoV-2 Utility Channel test setup\nA 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.\nTable 1 SARS-CoV-2 PCR cycling conditions and software settings in the Utility Channel software used to create the run template\nSample type Alcohol-based swab (400 µL input)\nChannels 1 2:SARS-CoV-2 E-gene 3 4 IC\nRFI NA 1.5 NA NA Predefined\nPCR cycling conditions UNG incubation Pre-PCR step 1st measurement 2nd measurement Cooling\nNumber of cycles Predefined 1 5 45 Predefined\nNumber of steps 3 2 2\nTemperature 55 °C; 60 °C; 65 °C 95 °C; 55 °C 91 °C; 58 °C\nHold time 120 s; 360 s; 240 s 5 s; 30 s 5 s; 25 s\nData acquisition None End of each cycle End of each cycle\nIC: 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 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