Simulation of SARS-CoV-2-positive oropharyngeal swabs and storage
To ensure reproducibility with a defined amount of virus in the samples, SARS-CoV-2-positive oropharyngeal swabs were simulated. For this purpose, healthy laboratory staff members swabbed themselves using two common swab types: wet eSwab with amies medium (Copan, Brescia, IT, no. 490CE) and dry FLOQSwabs (Copan, Brescia, IT, no. 552c). The following steps were performed in a BSL-3 laboratory. After self-testing, the swabs were transferred into 2-ml microcentrifuge tubes (Eppendorf, Hamburg, DE) containing 500 µl of cell culture supernatant with a total of 5x103 PFU of SARS-2-CoV (strain BetaCoV/Germany/BavPat1/2020, kindly provided by the Institute for Microbiology of the German Armed Forces). Swabs were wiped around in the virus suspension for approximately 10 seconds and subsequently transferred into their associated storage vessels (transport medium or dry tube). Swabs in storage vessels were either stored at room temperature (RT) or in a refrigerator at 4 °C. Samples for real-time PCR and virus cultivation were taken immediately, after two and four days of storage. For each time point and each swab type, samples were prepared and analysed in biological triplicate.
Real-time PCR analysis
For nucleic acid extraction the QIAamp® Viral RNA Mini Kit (Qiagen, Hilden, DE) was used. Briefly, dry swabs were vortexed in 1 ml of PBS and 140 µl were transferred into 560 µl of AVL buffer. For wet swabs 140 µl of storage medium were used for extraction. Viral and cellular nucleic acids were analysed using an in-house real-time PCR as described by Michel et al. [2]. In a duplex assay, both the SARS-CoV-2 orf1ab gene and the cellular MYC gene were targeted.
Virus cultivation
All samples were stored at -80°C until virus cultivation. Isolation of infectious SARS-CoV-2 virions was done on VeroE6 cells (Vero C1008; ECACC 85020206) which were cultivated in DMEM medium containing 10 % FCS, 1 % L-Gln, 100 U/ml of Penicillin and Streptomycin and 50 mg/ml of Normocin™ (Invivogen, San Diego, CA, USA). For SARS-CoV-2 infection, DMEM medium containing a reduced serum concentration of 2 % FCS was used. All infection experiments were performed in a BSL-3 laboratory. Briefly, 50 µl of each simulated swab sample were diluted by addition of 150 µl of infection medium. The diluted sample was used to infect one well of VeroE6 cells cultivated in 24-well cell culture plates. All samples were tested in triplicate. After 1 h of incubation at 37°C and 5 % CO2 to allow adhesion of SARS-CoV-2, the diluted sample was removed and the cells were washed once in PBS, before adding 500 µl of fresh medium. Subsequently, the cells were incubated at 37°C and 5 % CO2 for three days. At day 3 post infection (p.i.), cells were examined for signs of cytopathic effect (CPE). Furthermore, supernatant from two of the three replicate wells was harvested on day 3 and subjected to RNA extraction and real-time PCR analysis. The remaining replicate was cultivated until day 7 p.i. before it was processed equally.
Virus titration (TCID50)
A total of 2x104 VeroE6 cells per well in 100 µl of medium (DMEM + 10 % FCS + 2 mM L-Gln) were seeded in 96-well plates and incubated overnight at 37°C, 5% CO2. Samples were serially diluted in medium (10-1 to 10-10) and 100 µl of each dilution were added in eight replicates to the cells. After incubation for five days at 37°C, 5% CO2 the CPE was analysed by light microscopy and the tissue culture infectious dose 50 (TCID50) was calculated.