Journal
JOURNAL OF VIROLOGICAL METHODS
Volume 292, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.jviromet.2021.114115
Keywords
SARS-CoV-2; Droplet-digital PCR; Quantitative assays; Coronavirus; Viral transcription; replication
Funding
- Emergency COVID-19 Research Seed Funding of the University of California [R00RG3113]
- National Institute of Diabetes and Digestive and Kidney Diseases at the NIH [R01DK108349, R01DK120387]
- National Institute of Allergy and Infectious Diseases at the NIH [R01AI132128]
- UCSF/GIVI Center for AIDS Research (CFAR) [P30 AI027763, A120163]
- California HIV/AIDS Research Program [BB19-SF-009]
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The study developed and validated a novel panel of sensitive, quantitative RT-ddPCR assays designed to target regions spanning the genome of SARS-CoV-2, providing insights into the gene expression dynamics of the virus.
A hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SARCoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of sensitive, quantitative RT-ddPCR assays designed to target regions spanning the genome of SARS-CoV-2. Our assays target untranslated regions (5?, 3?) as well as different coding regions, including non-structural genes that are only found in full length (genomic) RNA and structural genes that are found in genomic as well as different subgenomic RNAs. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 gene expression and may also inform the development of improved diagnostic tools and therapeutics.
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