SARS-CoV-2 Viral RNA Detection

Viral RNA was isolated from homogenized tissues using the PureLink RNA minikit (Invitrogen). A one-step RT-PCR kit (Bio-Rad) was used to detect the viral RNA using Applied Biosystems QuantStudio 12K Flex real-time PCR system with the following cycling protocol: reverse transcription at 50°C for 10 min; hot start at 95°C for 10 min; and 40 cycles of denaturation at 95°C for 10 s and annealing at 60°C for 30 s. The primer sequences were CoV2-S_19F (5′ -GCTGAACATGTCAACAACTC- 3′) and CoV2-S_143R (5′ -GCAATGATGGATTGACTAGC- 3′), which were designed to target a 125 bp region of the SARS-CoV-2 spike protein (38 (link)). The standard samples were serial 10-fold dilutions of a known copy number of the HKU1 virus. The results were normalized and expressed as genome equivalent copies per gram of tissue.

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Dong W., Mead H., Tian L., Park J.G., Garcia J.I., Jaramillo S., Barr T., Kollath D.S., Coyne V.K., Stone N.E., Jones A., Zhang J., Li A., Wang L.S., Milanes-Yearsley M., Torrelles J.B., Martinez-Sobrido L., Keim P.S., Barker B.M., Caligiuri M.A, & Yu J. (2022). The K18-Human ACE2 Transgenic Mouse Model Recapitulates Non-severe and Severe COVID-19 in Response to an Infectious Dose of the SARS-CoV-2 Virus. Journal of Virology, 96(1), e00964-21.

Publication 2021

PureLink RNA minikit one-step RT-PCR kit QuantStudio 12K Flex

Dilutions Genome Primer Reverse transcription Rt pcr Sars cov 2 spike protein Tissue Viral rna Virus

Corresponding Organization :

Other organizations : City Of Hope National Medical Center, Northern Arizona University, Texas Biomedical Research Institute, Medical College of Wisconsin, The Ohio State University

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Variable analysis

independent variables

Primer sequences: CoV2-S_19F and CoV2-S_143R

dependent variables

Viral RNA detection and quantification

control variables

Reverse transcription at 50°C for 10 min
Hot start at 95°C for 10 min
40 cycles of denaturation at 95°C for 10 s and annealing at 60°C for 30 s
Sample type: Homogenized tissues
RNA extraction method: PureLink RNA minikit (Invitrogen)
RT-PCR kit: One-step RT-PCR kit (Bio-Rad)
Real-time PCR system: Applied Biosystems QuantStudio 12K Flex

controls

Positive control: Serial 10-fold dilutions of a known copy number of the HKU1 virus

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