PMC:7574920 / 45521-47229
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7574920","sourcedb":"PMC","sourceid":"7574920","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7574920","text":"RNA isolation and RT-qPCR\nThe standard diagnostic pipeline of the hospital laboratory was as follows: RNA was isolated from nasopharyngeal and oropharyngeal swab specimens using QIAGEN kits (QIAGEN, Hilden, Germany); either automated on the QIASymphony (DSP Virus/Pathogen Mini Kits) or QIAcube (QIAamp Viral RNA Mini Kits) devices or manually (QIAamp Viral RNA Mini Kits). Please note that the QiaCube uses a sample volume of 140 μl and an elution volume of 100 μl, whereas the QiaSymphony uses a sample volume of 200 μl and an elution volume of 115 μl. RT-qPCR for the quantification of the SARS-CoV-2 viral genome was performed using kits and reagents from TIB MOLBIO Syntheselabor, Berlin, Germany. The kits were used according to the manufacturer’s instruction and contained the primer/probe sets developed based on the published Sarbeco primer set (15). Per 20-μl reaction, the master mix contained 5.4 μl of RNAse free water, 4.0 μl of LightCycler Multiplex RNA Virus Master (Roche, Basel, Switzerland), 0.5 μl of LightMix Modular SARS and Wuhan CoV E gene (cat. no. 53-0776-96; TIB MOLBIOL Syntheselabor GmbH, Berlin, Germany) or LightMix Modular SARS and Wuhan CoV N gene (cat. no. 53-0775-96; TIB MOLBIOL), 0.5 μl of LightMix Modular EAV RNA Extraction Control (cat. no. 66-0909-96; TIB MOLBIOL), and 0.1 μl of reverse transcriptase enzyme (LightCycler Multiplex RNA Virus Master, Roche, Basel, Switzerland). The master mix (10 μl) was distributed per reaction into 96-well plates, and 10 μl of purified RNA was added per well. The performance of the RT-qPCR was validated using a positive control for the E gene. A total of 103 molecules of E gene RNA per RT-qPCR reaction correspond to a CT ≈ 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