Quantitative Detection of SARS-CoV-2 Subgenomic RNA

Viral subgenomic mRNA (sgRNA) is transcribed in infected cells and is not packaged into virions. Thus, presence of sgRNA is indicative of active infection of a mammalian cell in samples. We therefore measure sgRNA in all BAL samples obtained targeting the E gene as previously described.^{19 (link),20 (link)} Briefly, five μl RNA was used in a one-step real-time RT-PCR assay to sgRNA (forward primer 5’- CGATCTCTTGTAGATCTGTTCTC-3’; reverse primer 5’- ATATTGCAGCAGTACGCACACA-3’; probe 5’-FAMACACTAGCCATCCTTACTGCGCTTCG-ZEN-IBHQ-3’) and using the Quantifast Probe RT-PCR kit (Qiagen) according to instructions of the manufacturer. In each run, standard dilutions of counted RNA standards were run in parallel to calculate copy numbers in the samples.

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Sulaiman I., Chung M., Angel L., Tsay J.C., Wu B.G., Yeung S.T., Krolikowski K., Li Y., Duerr R., Schluger R., Thannickal S.A., Koide A., Rafeq S., Barnett C., Postelnicu R., Wang C., Banakis S., Pérez-Pérez L., Shen G., Jour G., Meyn P., Carpenito J., Liu X., Ji K., Collazo D., Labarbiera A., Amoroso N., Brosnahan S., Mukherjee V., Kaufman D., Bakker J., Lubinsky A., Pradhan D., Sterman D.H., Weiden M., Hegu A., Evans L., Uyeki T.M., Clemente J.C., de Wit E., Schmidt A.M., Shopsin B., Desvignes L., Wang C., Li H., Zhang B., Forst C.V., Koide S., Stapleford K.A., Khanna K.M., Ghedin E, & Segal L.N. (2021). Microbial signatures in the lower airways of mechanically ventilated COVID19 patients associated with poor clinical outcome. Nature microbiology, 6(10), 1245-1258.

Publication 2021

Quantifast Probe RT-PCR kit

Assay Cell Dilutions Gene Infection Mammalian Primer Real time pcr Rt pcr Subgenomic mrna Virions

Corresponding Organization :

Other organizations : New York University, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Longhua Hospital Shanghai University of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, University of Washington, Centers for Disease Control and Prevention, Icahn School of Medicine at Mount Sinai, NYU Langone Health

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Presence of viral subgenomic mRNA (sgRNA)

control variables

Amount of RNA used in RT-PCR assay (5 μl)
Use of one-step real-time RT-PCR assay to detect sgRNA
Forward primer sequence: 5'- CGATCTCTTGTAGATCTGTTCTC-3'
Reverse primer sequence: 5'- ATATTGCAGCAGTACGCACACA-3'
Probe sequence: 5'-FAMACACTAGCCATCCTTACTGCGCTTCG-ZEN-IBHQ-3'
Use of Quantifast Probe RT-PCR kit (Qiagen)
Running standard dilutions of counted RNA standards in parallel to calculate copy numbers in the samples

controls

Positive control: Standard dilutions of counted RNA standards
Negative control: Not explicitly mentioned

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