Tagman fast virus 1 step master mix

Virus replication was analyzed via qRT-PCR, and viral RNA was isolated from the supernatant at indicated hours postinfection using the NucleoMag Vet Kit (Macherey Nagel) and a Kingfisher Flex Purification System (Thermo Fisher Scientific) according to the manufacturer’s guidelines. Extracted RNA was amplified using TagMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific). The following primers were used for detection of MERS-CoV [87 (link)]:
and SARS-CoV and SARS-CoV-2:
targeting the Envelope gene of SARS-CoV-2 (MN908947.3). The primers were adapted from Corman and colleagues [88 ]. A serial dilution of in vitro transcribed (IVT) MERS-CoV RNA (kindly provided by Marcel Müller and Christian Drosten) [87 (link)] and RdRp-E-N RNA mixture derived from a SARS-CoV-2 synthetic construct (MT108784) was included to determine the genome copy number [89 (link)]. Five IVT RNA preparations were produced from 5 different DNA fragments to cover the regions used for real-time RT-qPCR methods for the detection of SARS-CoV-2 and SARS-CoV viral RNA. Measurements and analysis were performed with the Applied Biosystems 7500 Fast Dx Real-Time PCR Systems and associated software (Applied Biosystems, Foster City, California, USA).

Trizol (Sigma‐Aldrich) in a ratio of 1:3 was used to inactivate potential virus in nasal lavage samples (50 μl) from SARS‐CoV‐2‐infected K18‐hACE2 mice. For lung and spleen, PBS was added to each sample (1 g/ml) and pestles were used to crush the organs. Thereafter, the samples were centrifuged (5 min at 7,000 rpm) and 50 μl of each lung or spleen sample was added to Trizol (1:3). Total RNA was extracted using the Direct‐zol RNA Miniprep kit (Zymo Research) according to the manufacturer's instructions. Viral RNA were thereafter measured by quantitative real‐time polymerase chain reaction (qRT‐PCR) using TagMan Fast Virus 1‐Step master mix (Thermo Fisher Scientific) with primers and probe for the SARS‐CoV‐2 E gene.
Forward: 5′‐ ACAGGTACGTTAATAGTTAATAGCGT ‐3′
Reverse: 5′‐ ATATTGCAGCAGTACGCACACA ‐3′
Probe: FAM‐ ACACTA GCC ATC CTT ACT GCG CTT CG MGB
For lung and spleen samples, mouse ACTB mix (Thermo Fisher Scientific) was used as endogenous control. The PCR reaction was performed using a capillary Roche LightCycler 2.0 system.