Qiacube ht automated system

Swabs from hamsters were collected as described above. Cage and bedding material were sampled with prewetted swabs in 1 mL of DMEM supplemented with 200 U/mL penicillin and 200 μg/mL streptomycin. Then, 140 μL was utilized for RNA extraction using the QIAamp Viral RNA Kit (Qiagen) using QIAcube HT automated system (Qiagen) according to the manufacturer’s instructions with an elution volume of 150 μL. Sub-genomic (sg) viral RNA and genomic (g) was detected by qRT-PCR [27 , 61 ]. Five μL RNA was tested with TaqMan- Fast Virus One-Step Master Mix (Applied Biosystems) using QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems) according to instructions of the manufacturer. Ten-fold dilutions of SARS-CoV-2 standards with known copy numbers were used to construct a standard curve and calculate copy numbers/mL.

Swabs from hamsters were collected as described above. Then, 140 μL was utilized for RNA extraction using the QIAamp Viral RNA Kit (Qiagen) using QIAcube HT automated system (Qiagen) according to the manufacturer’s instructions with an elution volume of 150 μL. For tissues, RNA was isolated using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions and eluted in 60 μL. Sub-genomic (sg) viral RNA and genomic (g) was detected by qRT-PCR^{52 (link),53 (link)}. RNA was tested with TaqMan^™ Fast Virus One-Step Master Mix (Applied Biosystems) using QuantStudio 6 or 3 Flex Real-Time PCR System (Applied Biosystems). SARS-CoV-2 standards with known copy numbers were used to construct a standard curve and calculate copy numbers/mL or copy numbers/g.

Swabs from hamsters were collected as described above. Then, 140 μL was utilized for RNA extraction using the QIAamp Viral RNA Kit (Qiagen) using QIAcube HT automated system (Qiagen) according to the manufacturer’s instructions with an elution volume of 150 μL. For tissues, RNA was isolated using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions and eluted in 60 μL. Sub-genomic (sg) and genomic (g) viral RNA was detected by qRT-PCR [54 , 55 ]. RNA was tested with TaqMan^™ Fast Virus One-Step Master Mix (Applied Biosystems) using QuantStudio 3 Flex Real-Time PCR System (Applied Biosystems). SARS-CoV-2 standards with known copy numbers were used to construct a standard curve and calculate copy numbers/mL or copy numbers/g. The detection limit for the assay was 10 copies/reaction, and samples below this limit were considered negative.

Swabs from hamsters were collected as described above. Cage and bedding material was sampled with prewetted swabs in 1 mL of DMEM supplemented with 200 U/mL penicillin and 200 μg/mL streptomycin. Then, 140 μL was utilized for RNA extraction using the QIAamp Viral RNA Kit (Qiagen) using QIAcube HT automated system (Qiagen) according to the manufacturer’s instructions with an elution volume of 150 μL. Sub-genomic (sg) viral RNA and genomic (g) was detected by qRT-PCR (Corman et al., 2020a ; Corman et al., 2020b ). Five μL RNA was tested with TaqMan^™ Fast Virus One-Step Master Mix (Applied Biosystems) using QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems) according to instructions of the manufacturer. Ten-fold dilutions of SARS-CoV-2 standards with known copy numbers were used to construct a standard curve and calculate copy numbers/mL.

Samples were collected with prewetted swabs in 1 mL of DMEM supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin. Then, 140 μL was utilized for RNA extraction using the QIAamp Viral RNA Kit (Qiagen) using QIAcube HT automated system (Qiagen) according to the manufacturer's instructions with an elution volume of 150 μL. Tissues (up to 30 mg) were homogenized in RLT buffer and RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer's instructions. Viral RNA was detected by qRT-PCR [50 (link)]. Five μL RNA was tested with the Rotor-GeneTM probe kit (Qiagen) according to instructions of the manufacturer. Ten-fold dilutions of SARS-CoV-2 standards with known copy numbers were used to construct a standard curve.

Swabs from hamsters were collected as described above. Then, 140 µL was utilized for RNA extraction using the QIAamp Viral RNA Kit (Qiagen, Hilden Germany) using QIAcube HT automated system (Qiagen) according to the manufacturer’s instructions with an elution volume of 150 µL. Sub-genomic (sg) viral RNA and genomic (g) was detected by qRT-PCR [26 (link)]. Then, 5 μL RNA was tested with TaqMan™ Fast Virus One-Step Master Mix (Applied Biosystems, Foster City, CA, USA) using QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems) according to instructions of the manufacturer. Ten-fold dilutions of SARS-CoV-2 standards with known copy numbers were used to construct a standard curve and calculate copy numbers/mL.

Swabs from hamsters were collected as described above. 140 μL was utilized for RNA extraction using the QIAamp Viral RNA Kit (Qiagen) using QIAcube HT automated system (Qiagen) according to the manufacturer’s instructions with an elution volume of 150 μL. For tissues, RNA was isolated using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions and eluted in 60 μL. Sub-genomic (sg) and genomic (g) viral RNA were detected by qRT-PCR [49 ]. RNA was tested with TaqMan^™ Fast Virus One-Step Master Mix (Applied Biosystems) using QuantStudio 6 or 3 Flex Real-Time PCR System (Applied Biosystems). SARS-CoV-2 standards with known copy numbers were used to construct a standard curve and calculate copy numbers/mL or copy numbers/g. Limit of detection = 10 copies/rxn.